Sphingosine kinase enzyme

ABSTRACT

The present invention relates generally to novel protein molecules and to derivatives, analogues, chemical equivalents and mimetics thereof capable of modulating cellular activity and, in particular, modulating cellular activity via the modulation of signal transduction. More particularly, the present invention relates to human sphingosine kinase and to derivatives, analogues, chemical equivalents and mimetics thereof. The present invention also contemplates genetic sequences encoding said protein molecules and derivatives, analogues, chemical equivalents and mimetics thereof. The molecules of the present invention are useful in a range of therapeutic, prophylactic and diagnostic applications.

RELATED APPLICATIONS

This application is a division of parent application Ser. No. 10/642,289filed Aug. 18, 2003, itself a divisional of grandparent application09/959,897, nationalized Nov. 13, 2001, now allowed, which itself is thenational stage under 37 USC 371 of International applicationPCT/AU00/00457, filed May 12, 2000, which designated the United Statesand was published under PCT Article 21(2) in the English language.

FIELD OF THE INVENTION

The present invention relates generally to novel protein molecules andto derivatives, analogues, chemical equivalents and mimetics thereofcapable of modulating cellular activity and, in particular, modulatingcellular activity via the modulation of signal transduction. Moreparticularly, the present invention relates to human sphingosine kinaseand to derivatives, analogues, chemical equivalents and mimeticsthereof. The present invention also contemplates genetic sequencesencoding said protein molecules and derivatives, analogues, chemicalequivalents and mimetics thereof. The molecules of the present inventionare useful in a range of therapeutic, prophylactic and diagnosticapplications.

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to by author in thisspecification are collected alphabetically at the end of thedescription.

Sphingosine kinase is a key regulatory enzyme in a variety of cellularresponses. Its activity can affect inflammation, apoptosis and cellproliferation, and thus it is an important target for therapeuticintervention.

Sphingosine-1-phosphate is known to be an important second messenger insignal transduction (Meyer et al., 1997). It is mitogenic in variouscell types (Alessenko, 1998; Spiegel et al., 1998) and appears totrigger a diverse range of important regulatory pathways including;prevention of ceramide-induced apoptosis (Culliver et al., 1996),mobilisation of intracellular calcium by an IP₃-independant pathway,stimulation of DNA synthesis, activation of mitogen-activated protein(MAP) kinase pathway, activation of phospholipase D, and regulation ofcell motility (for reviews see Meyer et al., 1997; Spiegel et al., 1998;Igarashi., 1997).

Recent studies (Xia et al., 1998) have shown thatsphingosine-1-phosphate is an obligatory signalling intermediate in theinflammatory response of vascular endothelial cells to tumour necrosisfactor-α (TNFα). In spite of its obvious importance, very little isknown of the mechanisms that control cellular sphingosine-1-phosphatelevels. It is known that sphingosine-1-phosphate levels in the cell aremediated largely by its formation from sphingosine by sphingosinekinase, and to a lesser extent by its degradation by endoplasmicreticulum-associated sphingosine-1-phosphate lyase andsphingosine-1-phosphate phosphatase (Spiegel et al., 1998). Basal levelsof sphingosine-1-phosphate in the cell are generally low, but canincrease rapidly and transiently when cells are exposed to mitogenicagents. This response appears correlated with an increase in sphingosinekinase activity in the cytosol and can be prevented by addition of thesphingosine kinase inhibitory molecules N,N-dimethylsphingosine andDL-threo-dihydrosphingosine. This indicates that sphingosine kinase isan important molecule responsible for regulating cellularsphingosine-1-phosphate levels. This places sphingosine kinase in acentral and obligatory role in mediating the effects attributed tosphingosine-1-phosphate in the cell.

Accordingly, there is a need to identify and clone novel sphingosinekinase molecules to facilitate the progression towards the moresensitive control of intracellular signal transduction via, for example,the elucidation of the mechanism controlling the expression andenzymatic activity of sphingosine kinase thereby providing a platformfor the development of interventional therapies to regulate theexpression or activity of sphingosine kinase. In work leading up to thepresent invention the inventors have purified and cloned a novelsphingosine kinase molecule.

SUMMARY OF THE INVENTION

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

One aspect of the present invention provides an isolated nucleic acidmolecule or derivative or analogue thereof comprising a nucleotidesequence encoding or complementary to a sequence encoding a novelsphingosine kinase protein or a derivative or mimetic of saidsphingosine kinase protein.

Another aspect of the present invention provides an isolated nucleicacid molecule or derivative or analogue thereof comprising a nucleotidesequence encoding or complementary to a sequence encoding a humansphingosine kinase protein or a derivative or mimetic of saidsphingosine kinase protein.

Yet another aspect of the present invention provides a nucleic acidmolecule or derivative or analogue thereof comprising a nucleotidesequence encoding, or a nucleotide sequence complementary to anucleotide sequence encoding, an amino acid sequence substantially asset forth in SEQ ID NO:2 or a derivative or mimetic thereof or having atleast about 45% or greater similarity to at least 10 contiguous aminoacids in SEQ ID NO:2.

Still another aspect of the present invention contemplates a nucleicacid molecule or derivative or analogue thereof comprising a nucleotidesequence substantially as set forth in SEQ ID NO:1 or a derivativethereof or capable of hybridizing to SEQ ID NO:1 under low stringencyconditions.

Still yet another aspect of the present invention contemplates a nucleicacid molecule or derivative or analogue thereof comprising a nucleotidesequence substantially as set forth in SEQ ID NO:1 or a derivativethereof, or capable of hybridizing to SEQ ID NO:1 under low stringencyconditions and which encodes an amino acid sequence corresponding to anamino acid sequence set forth in SEQ ID NO:2 or a sequence having atleast about 45% similarity to at least 10 contiguous amino acids in SEQID NO:2.

A further aspect of the present invention contemplates a nucleic acidmolecule comprising a sequence of nucleotides substantially as set forthin SEQ ID NO:1.

Another further aspect of the present invention contemplates a genomicnucleic acid molecule or derivative or analogue thereof capable ofhybridizing to SEQ ID NO:1 or a derivative thereof under low stringencyconditions at 42° C.

Still another further aspect of the present invention provides a cDNAsequence comprising a sequence of nucleotides as set forth in SEQ IDNO:1 or a derivative or analogue thereof including a nucleotide sequencehaving similarity to SEQ ID NO:1.

Yet another further aspect of the present invention provides an aminoacid sequence set forth in SEQ ID NO:2 or a derivative, analogue orchemical equivalent or mimetic thereof as defined above or a derivativeor mimetic having an amino acid sequence of at least about 45%similarity to at least 10 contiguous amino acids in the amino acidsequence as set forth in SEQ ID NO:2 or a derivative or mimetic thereof.

Still yet another further aspect of the present invention is directed toan isolated protein selected from the list consisting of:

-   -   (i) A novel sphingosine kinase protein or a derivative,        analogue, chemical equivalent or mimetic thereof.    -   (ii) A human sphingosine kinase protein or a derivative,        analogue, chemical equivalent or mimetic thereof.    -   (iii) A protein having an amino acid sequence substantially as        set forth in SEQ ID NO:2 or a derivative or mimetic thereof or a        sequence having at least about 45% similarity to at least 10        contiguous amino acids in SEQ ID NO:2 or a derivative, analogue,        chemical equivalent or mimetic of said protein.    -   (iv) A protein encoded by a nucleotide sequence substantially as        set forth in SEQ ID NO:1 or a derivative or analogue thereof or        a sequence encoding an amino acid sequence having at least about        45% similarity to at least 10 contiguous amino acids in SEQ ID        NO:2 or a derivative, analogue, chemical equivalent or mimetic        of said protein.    -   (v) A protein encoded by a nucleic acid molecule capable of        hybridising to the nucleotide sequence as set forth in SEQ ID        NO:1 or a derivative or analogue thereof under low stringency        conditions and which encodes an amino acid sequence        substantially as set forth in SEQ ID NO:2 or a derivative or        mimetic thereof or an amino acid sequence having at least about        45% similarity to at least 10 contiguous amino acids in SEQ ID        NO:2.    -   (vi) A protein as defined in paragraphs (i) or (ii) or (iii)        or (iv) or (v) in a homodimeric form.    -   (vii) A protein as defined in paragraphs (i) or (ii) or (iii)        or (iv) or (v) in a heterodimeric form.

Another aspect of the present invention contemplates a method ofmodulating activity of sphingosine kinase in a mammal, said methodcomprising administering to said mammal a modulating effective amount ofan agent for a time and under conditions sufficient to increase ordecrease sphingosine kinase activity.

Still another aspect of the present invention contemplates a method ofmodulating cellular functional activity in a mammal said methodcomprising administering to said mammal an effective amount of an agentfor a time and under conditions sufficient to modulate the expression ofa nucleotide sequence encoding sphingosine kinase or sufficient tomodulate the activity of sphingosine kinase.

Yet another aspect of the present invention contemplates a method ofmodulating cellular functional activity in a mammal said methodcomprising administering to said mammal an effective amount ofsphingosine kinase or sphingosine kinase.

Still yet another aspect of the present invention relates to a method oftreating a mammal said method comprising administering to said mammal aneffective amount of an agent for a time and under conditions sufficientto modulate the expression of sphingosine kinase or sufficient tomodulate the activity of sphingosine kinase wherein said modulationresults in modulation of cellular functional activity.

A further aspect of the present invention relates to a method oftreating a mammal said method comprising administering to said mammal aneffective amount of sphingosine kinase or sphingosine kinase for a timeand under conditions sufficient to modulate cellular functionalactivity.

Yet another further aspect of the present invention relates to the useof an agent capable of modulating the expression of sphingosine kinaseor modulating the activity of sphingosine kinase in the manufacture of amedicament for the modulation of cellular functional activity.

A further aspect of the present invention relates to the use ofsphingosine kinase or sphingosine kinase in the manufacture of amedicament for the modulation of cellular functional activity.

Still yet another aspect of the present invention relates to agents foruse in modulating sphingosine kinase expression or sphingosine kinaseactivity wherein said modulation results in modulation of cellularfunctional activity.

Another aspect of the present invention relates to sphingosine kinase orsphingosine kinase for use in modulating cellular functional activity.

In a related aspect of the present invention, the mammal undergoingtreatment may be a human or an animal in need of therapeutic orprophylactic treatment.

In yet another further aspect the present invention contemplates apharmaceutical composition comprising sphingosine kinase, sphingosinekinase or an agent capable of modulating sphingosine kinase expressionor sphingosine kinase activity together with one or morepharmaceutically acceptable carriers and/or diluents. Sphingosinekinase, sphingosine kinase or said agent are referred to as the activeingredients.

Yet another aspect of the present invention contemplates a method fordetecting sphingosine kinase or sphingosine kinase mRNA in a biologicalsample from a subject said method comprising contacting said biologicalsample with an antibody specific for sphingosine kinase or sphingosinekinase mRNA or its derivatives or homologs for a time and underconditions sufficient for an antibody-sphingosine kinase orantibody-sphingosine kinase mRNA complex to form, and then detectingsaid complex.

Single and three letter abbreviations used throughout the specificationare defined in Table 1. TABLE 1 Single and three letter amino acidabbreviations Three-Letter Amino Acid Abbreviation One-letter SymbolAlanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp DCysteine Cys C Glutamine Gln Q Glutamic acid Glu E Glycine Gly GHistidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K MethionineMet M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine The TTryptophan Trp W Tyrosine Tyr Y Valine Val V Any residue Xaa X

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the ‘Sphingomyelin pathway’.

FIG. 2 is a graphical representation of anion exchange chromatography ofhuman sphingosine kinase. A, Anion exchange chromatography with QSepharose fast flow of the (NH₄)₂SO₄ precipitated fraction of the humanplacenta extract showing two peaks of sphingosine kinase activity.Extracts were applied in buffer A and sphingosine kinase activity (•)was eluted with a NaCl gradient of 0 to 1M (- - -). Protein eluted wasfollowed by absorbance at 280 nm (—).

FIG. 3 is a graphical representation of the purification of humanplacenta SKI by anion exchange chromatography. Purification on a Mono-Qcolumn is enhanced by reapplication and elution from the same column inthe presence of ATP. A, The fractions from the calmodulin-Sepharose 4bcolumn containing sphingosine kinase activity were pooled, desalted andapplied to the Mono-Q column in buffer A. B, Active fractions from theMono-Q column were desalted and reapplied to the Mono-Q column in bufferA with 1 mM ATP and 4mM MgCl₂. In both cases sphingosine kinase activity(•) was eluted with a NaCl gradient of 0 to 1M (- - -), with proteinelution followed by absorbance at 280 nm (—).

FIG. 4 is an image of SDS-PAGE of purified human placenta sphingosinekinase. The fraction from the Superdex 75 column containing the highestsphingosine kinase activity was applied to SDS-PAGE with silverstaining, yielding a single band of 45 kDa.

FIG. 5 is a graphical representation of preparative anion exchangechromatography. Only a single chromatographically identified sphingosinekinase isoform is present in HUVEC. Preparative anion exchangechromatography with HiTrap-Q columns of human placenta, HUVEC and TNFαtreated HUVEC extracts showing, in HUVEC, the presence of a singlesphingosine kinase peak that increases in activity following treatmentof cells with TNFα. Cells were harvested, lysed and the soluble extractsapplied to the HiTrap-Q column in buffer A. Total sphingosine kinaseactivity in human placenta, HUVEC and TNFα treated HUVEC extracts were51, 78 and 136 U/mg protein, respectively. Sphingosine kinase activity(□) was eluted with a NaCl gradient of 0 to 1M (- - -).

FIG. 6 is a schematic representation of the strategy used to clonesphingosine kinase (SPHK) from HUVEC.

FIG. 7 is a schematic representation of the nucleotide (nucleotides15-1187 of SEQ ID NO:1) and deduced amino acid (SEQ ID NO:2) sequencesand putative domain structure of human sphingosine kinase. A, cDNAnucleic acid sequence and the deduced amino acid sequence of hSK. Aminoacids are numbered from the first methionine residue. The stop codon isindicated by an asterisk. The sphingosine kinase-coding region is incapital letters (nucleotides 33-1187), while lower case letters denoteuntranslated and vector sequence. B, Schematic representation of humansphingosine kinase showing locations of the putative PKC and CKIIphosphorylation sites, a possible N-myristoylation site,calcium/calmodulin binding motifs and the region with similarity to theputative DGK catalytic domain.

FIG. 8 is a graphical representation of the expression and TNFαstimulation of human sphingosine kinase activity in HEK293 cells. HEK293cells transiently transfected with either empty pcDNA expression vectoralone (A) or pcDNA containing human sphingosine kinase cDNA (B).Transfected cells were either untreated or treated with TNFα for 10 min,harvested and sphingosine kinase activity in cell lysates determined.Data are means of duplicates and are representative of three independentexperiments.

FIG. 9 is a schematic representation of the sequence comparison of humansphingosine kinase with other known and putative sphingosine kinases.Comparison of the deduced human sphingosine kinase amino acid sequencewith the amino acid sequences of the murine (mSK1a (SEQ ID NOs:9-16) andmSK1b (SEQ ID NOs:17-24); Kohama et al., 1998) and S. cerevisiae (LCB4(SEQ ID NOs:25-32) and LCB5 (SEQ ID NOs:33-40); Nagiec et al., 1998)sphingosine kinases, and EST sequences of putative sphingosine kinasesfrom S. pombe (SEQ ID NOs:41-48) and C. elegans (SEQ ID NOs:49-56)(Genbank™ accession numbers Z98762 and Z66494, respectively). Althoughthe amino acid sequence similarity to human sphingosine kinase was high(36% identity), the A. thaliana putative sphingosine kinase sequence(Genbank™ accession number AL022603) gave relatively poor alignment and,for clarity, is not shown. The consensus sequence represents amino acidsthat are conserved in at least six of the seven aligned sequences, whileconservation of structurally similar amino acids are denoted with anasterisk. Multiple sequence alignment was performed with CLUSTALW, andpercentage identities to the human sphingosine kinase were determinedusing the GAP algorithm (Needleman & Wunsch, 1970).

FIG. 10 is an image of the expression of recombinant human sphingosinekinase in E. coli BL21. E. coli BL21 was transformed with the pGEX4-2Thuman sphingosine kinase expression construct and expression of theGST-SK fusion protein analyzed after induction with 100 μM IPTG. A,Sphingosine kinase activity in uninduced and IPTG induced E. coli BL21cell extracts. B, Coomassie stained SDS-PAGE gel showing GST-SK fusionprotein expression in E. coli cell lysates. C, Purity of the isolatedrecombinant human sphingosine kinase. The fraction from the Mono-Qcolumn containing sphingosine kinase activity was applied to SDS-PAGEwith silver staining yielding a single band of 45 kDa.

FIG. 11 is an image of the purification of recombinant human sphingosinekinase. Calmodulin Sepharose 4B allows the separation of active andinactive enzyme. The GST-SK fusion protein was partially purified usingglutathione-Sepharose 4B, cleaved by thrombin and applied to thecalmodulin-Sepharose 4B column in Buffer B containing 4 mM CaCl₂.Elution (1) of the active sphingosine kinase bound to the column wasperformed with Buffer A containing 2 mM EGTA and 1 M NaCl. A, SDS-PAGEanalysis of fractions eluted from the calmodulin-Sepharose 4B column. B,Sphingosine kinase activity (|) in column fractions showing most of therecombinant human sphingosine kinase protein did not bind to the columnand displayed no catalytic activity.

FIG. 12 is a graphical representation of the physico-chemical propertiesof the native and recombinant sphingosine kinases. A, pH optima. Theeffect of pH on SK activity was determined by assaying the activity overthe pH range of 4 to 11 in 50 mM buffers (sodium acetate, pH 4.0-5.0;Mes, pH 6.0-7.0; Hepes, pH 7.0-8.2; Tris/HCl, pH 8.2-10.0; Caps, pH10.0-11.0). B, pH stability. Data shown is the SK activity remainingafter preincubation of the enzymes at various pH at 4° C. for 5 h. C,Temperature stability. Data shown is the SK activity remaining afterpreincubation of the enzymes at various temperatures (4 to 80° C.) for30 min at pH 7.4 (50mM Tris/HCl containing 10% glycerol, 0.5 M NaCl and0.05% Triton X-100). D, Metal ion requirement. The various metal ions orEDTA were supplied in the assay mixture at a final concentration of 10mM. In all cases the maximum activities of the native (□ and filledbars) and recombinant (□ and open bars) sphingosine kinases werearbitrarily set at 100% and correspond to 2.65 kU and 7.43 kU,respectively. Data are means ±S.D.

FIG. 13 is a graphical representation of the substrate specificity andkinetics of the native and recombinant sphingosine kinases. A, Substratespecificity of the native (filled bars) and recombinant (open bars)sphingosine kinases with sphingosine analogues and other lipids suppliedat 100 μM in 0.25% Triton X-100. The rates of phosphorylation ofsphingosine by the native and recombinant Sks were arbitrarily set at100% and correspond to 2.65 kU and 7.43 kU, respectively. Activityagainst other potential substrates were expressed relative to theactivity against sphingosine. No phosphorylation was observed withDL-threo-dihydrosphingosine, N,N-dimethylsphingosine,N,N,N,-trimethylsphingosine, N-acetylsphingosine (C₂-ceramide),diacylglycerol (1,2-dioctanoyl-sn-glycerol and1,2-dioleoyl-sn-glycerol), and phosphatidylinositol. B, Substratekinetics of the recombinant human sphingosine kinase with sphingosine(□) and D-erythro-dihydrosphingosine (□) as substrates. C, Kinetics ifinhibition of the recombinant human sphingosine kinase withN,N,N-trimethylsphingosine at 5 μM (□) and 25 μM (□), and in the absenceof N,N,N-trimethylsphingosine (□). Inset: Lineweaver-Burk plot. Data aremeans ±S.D.

FIG. 14 is a graphical representation of the acidic phospholipids whichstimulate the activity of the native and recombinant sphingosinekinases. The effect of various phospholipids on the activity of thenative and recombinant sphingosine kinases were determined by assayingthe activity under standard conditions in the presence if thesephospholipids at 10 mol % of Triton X-100. The activities of the native(filled bars) and recombinant (open bars) sphingosine kinases in theabsence of phospholipids were arbitrarily set at 100% and correspond to2.65 kU and 7.43 kU, respectively. PC, phosphatidylcholine; PS,phosphatidylserine; PE, phosphatidylethanolamine; PIphosphatidylinositol; PA, phosphatidic acid. Data are means ±S.D.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated, in part, on the purification andcloning of a novel sphingosine kinase molecule. The identification ofthis novel molecule permits the identification and rational design of arange of products for use in therapy, diagnosis and antibody generation,for example for use in signal transduction. These therapeutic moleculesmay also act as either antagonists or agonists of sphingosine kinasefunction and will be useful, inter alia, in the modulation of cellularactivation in the treatment of disease conditions characterised byunwanted cellular activity.

Accordingly, one aspect of the present invention provides an isolatednucleic acid molecule or derivative or analogue thereof comprising anucleotide sequence encoding or complementary to a sequence encoding anovel sphingosine kinase protein or a derivative or mimetic of saidsphingosine kinase protein.

Reference to “sphingosine kinase” should be understood as a reference tothe molecule which is, inter alia, involved in the generation ofsphingosine-1-phosphate during the activation of the sphingosine kinasesignaling pathway. Reference to “sphingosine kinase” in italicized textshould be understood as a reference to the sphingosine kinase nucleicacid molecule. Reference to “sphingosine kinase” in non-italicized textshould be understood as a reference to the sphingosine kinase proteinmolecule.

More particularly, the present invention provides an isolated nucleicacid molecule or derivative or analogue thereof comprising a nucleotidesequence encoding of complementary to a sequence encoding a humansphingosine kinase protein or a derivative or mimetic of saidsphingosine kinase protein.

In a preferred embodiment, the present invention provides a nucleic acidmolecule or derivative or analogue thereof comprising a nucleotidesequence encoding, or a nucleotide sequence complementary to anucleotide sequence encoding, an amino acid sequence substantially asset forth in SEQ ID NO:2 or a derivative or mimetic thereof or having atleast about 45% or greater similarity to at least 10 contiguous aminoacids in SEQ ID NO:2.

The term “similarity” as used herein includes exact identity betweencompared sequences at the nucleotide or amino acid level. Where there isnon-identity at the nucleotide level, “similarity” includes differencesbetween sequences which result in different amino acids that arenevertheless related to each other at the structural, functional,biochemical and/or conformational levels. Where there is non-identity atthe amino acid level, “similarity” includes amino acids that arenevertheless related to each other at the structural, functional,biochemical and/or conformational levels. The percentage similarity maybe greater than 50% such as at least 70% or at least 80% or at least 90%or at least 95% or higher.

Another aspect of the present invention contemplates a nucleic acidmolecule or derivative or analogue thereof comprising a nucleotidesequence substantially as set forth in SEQ ID NO:1 or a derivativethereof, or capable of hybridizing to SEQ ID NO:1 under low stringencyconditions.

Reference herein to a low stringency includes and encompasses from atleast about 0% v/v to at least about 15% v/v formamide and from at leastabout 1M to at least about 2M salt for hybridization, and at least about1M to at least about 2M salt for washing conditions. Alternativestringency conditions may be applied where necessary, such as mediumstringency, which includes and encompasses from at least about 16% v/vto at least about 30% v/v formamide and from at least about 0.5M to atleast about 0.9M salt for hybridization, and at least about 0.5M to atleast about 0.9M salt for washing conditions, or high stringency, whichincludes and encompasses from at least about 31% v/v to at least about50% v/v formamide and from at least about 0.1M to at least about 0.15Msalt for hybridization, and at least about 0.01M to at least about 0.15Msalt for washing conditions. Stringency may be measured using a range oftemperature such as from about 40° C. to about 65° C. Particularlyuseful stringency conditions are at 42° C. In general, washing iscarried out at T_(m)=69.3 ±0.41 (G+C) % [19]=−12° C. However, the T_(m)of a duplex DNA decreases by 1° C. with every increase of 1% in thenumber of mismatched based pairs (Bonner et al (1973) J. Mol. Biol.,81:123).

Preferably, the present invention contemplates a nucleic acid moleculeor derivative or analogue thereof comprising a nucleotide sequencesubstantially as set forth in SEQ ID NO:1 or a derivative thereof orcapable of hybridizing to SEQ ID NO:1 under low stringency conditionsand which encodes an amino acid sequence corresponding to an amino acidsequence set forth in SEQ ID NO:2 or a sequence having at least about45% similarity to at least 10 contiguous amino acids in SEQ ID NO:2.

More particularly, the present invention contemplates a nucleic acidmolecule comprising a sequence of nucleotides substantially as set forthin SEQ ID NO:1.

The nucleic acid molecule according to this aspect of the presentinvention corresponds herein to human sphingosine kinase. Withoutlimiting the present invention to any one theory or mode of action, theprotein encoded by sphingosine kinase is a key element in thefunctioning of the sphingosine kinase-signaling pathway. Sphingosinekinase acts to facilitate the generation of the second messenger,sphingosine-1-phosphate, and may be activated by:

-   -   (a) post-translational modifications such as phosphorylation or        proteolytic cleavage;    -   (b) protein-protein interactions such as dimerization, and G        protein-coupled receptor mediated interactions;    -   (c) translocational events where the enzyme is targeted to an        environment that increases catalytic activity or allows access        to its substrate.

The expression product of the human sphingosine kinase nucleic acidmolecule is human sphingosine kinase. Sphingosine kinase is defined bythe amino acid sequence set forth in SEQ ID NO:2. The cDNA sequence forsphingosine kinase is defined by the nucleotide sequence set forth inSEQ ID NO:1. The nucleic acid molecule encoding sphingosine kinase ispreferably a sequence of deoxyribonucleic acids such as a cDNA sequenceor a genomic sequence. A genomic sequence may also comprise exons andintrons. A genomic sequence may also include a promoter region or otherregulatory regions.

Another aspect of the present invention contemplates a genomic nucleicacid molecule or derivative thereof capable of hybridizing to SEQ IDNO:1 or a derivative thereof under low stringency conditions at 42° C.

Reference herein to sphingosine kinase and sphingosine kinase should beunderstood as a reference to all forms of human sphingosine kinase andsphingosine kinase, respectfully, including, for example, any peptideand cDNA isoforms which arise from alternative splicing of sphingosinekinase mRNA, mutants or polymorphic variants of sphingosine kinase orsphingosine kinase, the post-translation modified form of sphingosinekinase or the non-post-translation modified form of sphingosine kinase.To the extent that it is not specified, reference herein to sphingosinekinase and sphingosine kinase includes reference to derivatives,analogues, chemical equivalents and mimetics thereof.

The protein and/or gene is preferably from the human. However, theprotein and/or gene may also be isolated from other animal or non-animalspecies.

Derivatives include fragments, parts, portions, mutants, variants andmimetics from natural, synthetic or recombinant sources including fusionproteins. Parts or fragments include, for example, active regions ofsphingosine kinase. Derivatives may be derived from insertion, deletionor substitution of amino acids. Amino acid insertional derivativesinclude amino and/or carboxylic terminal fusions as well asintrasequence insertions of single or multiple amino acids. Insertionalamino acid sequence variants are those in which one or more amino acidresidues are introduced into a predetermined site in the proteinalthough random insertion is also possible with suitable screening ofthe resulting product. Deletional variants are characterized by theremoval of one or more amino acids from the sequence. Substitutionalamino acid variants are those in which at least one residue in thesequence has been removed and a different residue inserted in its place.An example of substitutional amino acid variants are conservative aminoacid substitutions. Conservative amino acid substitutions typicallyinclude substitutions within the following groups: glycine and alanine;valine, isoleucine and leucine; aspartic acid and glutamic acid;asparagine and glutamine; serine and threonine; lysine and arginine; andphenylalanine and tyrosine. Additions to amino acid sequences includingfusions with other peptides, polypeptides or proteins.

Chemical and functional equivalents of sphingosine kinase or sphingosinekinase should be understood as molecules exhibiting any one or more ofthe functional activities of sphingosine kinase or sphingosine kinaseand may be derived from any source such as being chemically synthesizedor identified via screening processes such as natural product screening.

The derivatives of sphingosine kinase include fragments havingparticular epitopes or parts of the entire sphingosine kinase proteinfused to peptides, polypeptides or other proteinaceous ornon-proteinaceous molecules.

Analogues of sphingosine kinase contemplated herein include, but are notlimited to, modification to side chains, incorporating of unnaturalamino acids and/or their derivatives during peptide, polypeptide orprotein synthesis and the use of crosslinkers and other methods whichimpose conformational constraints on the proteinaceous molecules ortheir analogues.

Derivatives of nucleic acid sequences may similarly be derived fromsingle or multiple nucleotide substitutions, deletions and/or additionsincluding fusion with other nucleic acid molecules. The derivatives ofthe nucleic acid molecules of the present invention includeoligonucleotides, PCR primers, antisense molecules, molecules suitablefor use in co-suppression and fusion of nucleic acid molecules.Derivatives of nucleic acid sequences also include degenerate variants.

Examples of side chain modifications contemplated by the presentinvention include modifications of amino groups such as by reductivealkylation by reaction with an aldehyde followed by reduction withNaBH₄; amidination with methylacetimidate; acylation with aceticanhydride; carbamoylation of amino groups with cyanate;trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonicacid (TNBS); acylation of amino groups with succinic anhydride andtetrahydrophthalic anhydride; and pyridoxylation of lysine withpyridoxal-5-phosphate followed by reduction with NaBH₄.

The guanidine group of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation viaO-acylisourea formation followed by subsequent derivitisation, forexample, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylationwith iodoacetic acid or iodoacetamide; performic acid oxidation tocysteic acid; formation of a mixed disulphides with other thiolcompounds; reaction with maleimide, maleic anhydride or othersubstituted maleimide; formation of mercurial derivatives using4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid,phenylmercury chloride, 2-chloromercuri-4-nitrophenol and othermercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation withN-bromosuccinimide or alkylation of the indole ring with2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residueson the other hand, may be altered by nitration with tetranitromethane toform a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may beaccomplished by alkylation with iodoacetic acid derivatives orN-carboethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives duringprotein synthesis include, but are not limited to, use of norleucine,4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid,6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine,ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid,2-thienyl alanine and/or D-isomers of amino acids. A list of unnaturalamino acid contemplated herein is shown in Table 2. TABLE 2Non-conventional amino acid Code α-aminobutyric acid Abuα-amino-α-methylbutyrate Mgabu aminocyclopropane-carboxylate Cproaminoisobutyric acid Aib aminonorbornyl-carboxylate Norbcyclohexylalanine Cha cyclopentylalanine Cpen D-alanine Dal D-arginineDarg D-aspartic acid Dasp D-cysteine Dcys D-glutamine Ggln D-glutamicacid Dglu D-histidine Dhis D-isoleucine Dile D-leucine Dleu D-D-lysineDlys D-methionine Dmet D-ornithine Dorn D-phenylalanine Dphe D-prolineDpro D-serine Dser D-threonine Dthr D-tryptophan Dtrp D-tyrosine DtyrD-valine Dval D-α-methylalanine Dmala D-α-methylarginine DmargD-α-methylasparaginine Dmasn D-α-methylasparate Dmasp D-α-methylcysteineDmcys D-α-methylglutamine Dmgln D-α-methylhistidine DmhisD-α-methylisoleucine Dmile D-α-methylleucine Dmleu D-α-methyllysineDmlys D-α-methylmethionine Dmmet D-α-methylornithine DmornD-α-methylphenylalanine Dmphe D-α-methylproline Dmpro D-α-methylserineDmser D-α-methylthreonine Dmthr D-α-methyltryptophan DmtrpD-α-methyltyrosine Dmty D-α-methylvaline Dmval D-N-methylalanine DnmalaD-N-methylarginine Dnmarg D-N-methylasparagine Dnmasn D-N-methylasparateDnmasp D-N-methylcysteine Dnmcys D-N-methylglutamine DnmglnD-N-methylglutamate Dnmglu D-N-methylhistidine DnmhisD-N-methylisoleucine Dnmile D-N-methylleucine Dnmleu D-N-methyllysineDnmlys N-methylcyclohexylalanine Nmchexa D-N-methylornithine DnmornN-methylglycine Nala N-methylaminoisobutyrate NmaibN-(1-methylpropyl)glycine Nile N-(2-methylpropyl)glycine NleuD-N-methyltryptophan Dnmtrp D-N-methyltyrosine Dnmtyr D-N-methylvalineDnmval γ-aminobutyric acid Gabu L-t-butylglycine Tbug L-ethylglycine EtgL-homophenylalanine Hphe L-α-methylarginine Marg L-α-methylasparate HaspL-α-methylcysteine Mcys L-α-methylglutamine Mgln L-α-methylhistidineMhis L-α-methylisoleucine Mile L-α-methylleucine MleuL-α-methylmethionine Mmet L-α-methylnorvaline MnvaL-α-methylphenylalanine Mphe L-α-methylserine Mser L-α-methyltryptphanMtrp L-α-methylvaline Mval N-(N-2,2diphenylethyl)carbamymethyl)glycineNnbhm 1-carboxy-1(2,2,-diphenyl-ethylamino)cyclopropane NmbcL-N-methylalanine Nmala L-N-methylarginine Nmarg L-N-methylasparagineNmasn L-N-methylasparatic acid Nmasp L-N-methylcysteine NmcysL-N-methylglutamine Nmgln L-N-methylglutamic acid Nmglu ChexaL-N-methylhistidine Nmhis L-N-methylisoleucine Nmile L-N-methylleucineNmleu L-N-methyllysine Nmlys L-N-methylmethionine NmmetL-N-methylnorleucine Nmnle L-N-methylnorvaline Nmnva L-N-methylornithineNmorn L-N-methylphenylalanine Nmphe L-N-methylproline NmproL-N-methylserine Nmser L-N-methylthreonine Nmthr L-N-methyltryptophanNmtrp L-N-methyltyrosine Nmtyr L-N-methylvaline NmvalL-N-methylethylglycine Nmetg L-N-methyl-t-butylglycine NmtbugL-norleucine Nle L-norvaline Nva α-methyl-aminoisobutyrate Maibα-methyl-γ-aminobutyrate Mgabu α-methylcyclohexylalanine Mchexaα-methylcylcopentylalanine Mcpen α-methyl-α-napthylalanine Manapα-methylpenicilamine Mpen N-(4-aminobutyl)glycine NgluN-(2-aminoethyl)glycine Naeg N-(3-aminopropyl)glycine NornN-amino-α-methylbutyrate Nmaabu α-napthylalanine Anap N-benylglycineNphe N-(2-carbamylethyl)glycine Ngln N-(carbamymethyl)glycine NasnN-(2-carboxyethyl)glycine Nglu N(carboxymethyl)glycine NaspN-cyclobutylglycine Ncbut N-cycloheptylglycine Nchep N-cyclohexyglycineNehex N-cyclodecylglycine Ncdec N-cyclododecylglycine NcdodN-cyclooctylglycine Ncoct N-cyclopropylglycine NcproN-cycloundecylglycine Ncund N-(2,2-diphenylethyl)glycine NbhmN-(3,3-diphenylpropyl)glycine Nbhe N-(3-guanidinopropyl)glycine NargN-(1-hydroxyethyl)glycine Nthr N-(hydroxyethyl)glycine NserN-(imidazoylethyl)glycine Nhis N-(3-indolylethyl)glycine NhtrpN-methyl-γ-aminobutyrate Nmgabu D-N-methylmethionine DnmmetN-methylcyclopentylalanine Nmcpen D-N-methylphenylalanine DnmpheD-N-methylproline Dnmpro D-N-methylserine Dnser D-N-methylthreonineDnmthr N-(1-methylethyl)glycine Nval N-methyla-napthylalanine NmanapN-methylpencillamine Nmpen N-(p-hydroxyphenyl)glycine NhtyrN-(thiomethyl)glycine Ncys penicilamine Pen L-α-methylalanine MalaL-α-methylasparagine Masn L-α-methyl-t-butylglycine MtbugL-methylethylglycine Metg L-α-methylglutamate MgluL-α-methylhomophenylalanine Mhphe N-(2-methylthioethyl)glycine NmetL-α-methyllysine Mlys L-α-methylnorleucine Mnle L-α-methylornithine MornL-α-methylproline Mpro L-α-methylthreonine Mthr L-α-methyltyrosine MtyrL-N-methylhomophenylalanine NmhpheN-(N-(3,3-diphenylpropyl)carbamylmethyl)glycine Nnbhe

Crosslinkers can be used, for example, to stabilize 3D conformations,using homo-bifunctional crosslinkers such as the bifunctional imidoesters having (CH₂)_(n) spacer groups with n=1 to n=6, glutaraldehyde,N-hydroxysuccinimide esters and hetero-bifunctional reagents whichusually contain an amino-reactive moiety such as N-hydroxysuccinimideand another group specific-reactive moiety.

The nucleic acid molecule of the present invention is preferably inisolated form or ligated to a vector, such as an expression vector. By“isolated” is meant a nucleic acid molecule having undergone at leastone purification step and this is conveniently defined, for example, bya composition comprising at least about 10% subject nucleic acidmolecule, preferably at least about 20%, more preferably at least about30%, still more preferably at least about 40-50%, even still morepreferably at least about 60-70%, yet even still more preferably 80-90%or greater of subject nucleic acid molecule relative to other componentsas determined by molecular weight, encoding activity, nucleotidesequence, base composition or other convenient means. The nucleic acidmolecule of the present invention may also be considered, in a preferredembodiment, to be biologically pure.

The term “protein” should be understood to encompass peptides,polypeptides and proteins. The protein may be glycosylated orunglycosylated and/or may contain a range of other molecules fused,linked, bound or otherwise associated to the protein such as aminoacids, lipids, carbohydrates or other peptides, polypeptides orproteins. Reference hereinafter to a “protein” includes a proteincomprising a sequence of amino acids as well as a protein associatedwith other molecules such as amino acids, lipids, carbohydrates or otherpeptides, polypeptides or proteins.

In a particularly preferred embodiment, the nucleotide sequencecorresponding to sphingosine kinase is a cDNA sequence comprising asequence of nucleotides as set forth in SEQ ID NO:1 or a derivative oranalogue thereof including a nucleotide sequence having similarity toSEQ ID NO:1.

A derivative of a nucleic acid molecule of the present invention alsoincludes a nucleic acid molecule capable of hybridizing to a nucleotidesequence as set forth in SEQ ID NO:1 under low stringency conditions.Preferably, low stringency is at 42° C.

The nucleic acid molecule may be ligated to an expression vector capableof expression in a prokaryotic cell (e.g. E. coli) or a eukaryotic cell(e.g. yeast cells, fungal cells, insect cells, mammalian cells or plantcells). The nucleic acid molecule may be ligated or fused or otherwiseassociated with a nucleic acid molecule encoding another entity such as,for example, a signal peptide. It may also comprise additionalnucleotide sequence information fused, linked or otherwise associatedwith it either at the 3′ or 5′ terminal portions or at both the 3′ and5′ terminal portions. The nucleic acid molecule may also be part of avector, such as an expression vector. The latter embodiment facilitatesproduction of recombinant forms of sphingosine kinase which forms areencompassed by the present invention.

The present invention extends to the expression product of the nucleicacid molecules as hereinbefore defined.

The expression product is sphingosine kinase having an amino acidsequence set forth in SEQ ID NO:2 or is a derivative, analogue orchemical equivalent or mimetic thereof as defined above or is aderivative or mimetic having an amino acid sequence of at least about45% similarity to at least 10 contiguous amino acids in the amino acidsequence as set forth in SEQ ID NO:2 or a derivative or mimetic thereof.

Another aspect of the present invention is directed to an isolatedprotein selected from the list consisting of:

-   -   (i) A novel sphingosine kinase protein or a derivative,        analogue, chemical equivalent or mimetic thereof.    -   (ii) A human sphingosine kinase protein or a derivative,        analogue, chemical equivalent or mimetic thereof.    -   (iii) A protein having an amino acid sequence substantially as        set forth in SEQ ID NO:2 or a derivative or mimetic thereof or a        sequence having at least about 45% similarity to at least 10        contiguous amino acids in SEQ ID NO:2 or a derivative, analogue,        chemical equivalent or mimetic of said protein.    -   (iv) A protein encoded by a nucleotide sequence substantially as        set forth in SEQ ID NO:1 or a derivative or analogue thereof or        a sequence encoding an amino acid sequence having at least about        45% similarity to at least 10 contiguous amino acids in SEQ ID        NO:2 or a derivative, analogue, chemical equivalent or mimetic        of said protein.    -   (v) A protein encoded by a nucleic acid molecule capable of        hybridizing to the nucleotide sequence as set forth in SEQ ID        NO:1 or a derivative or analogue thereof under low stringency        conditions and which encodes an amino acid sequence        substantially as set forth in SEQ ID NO:2 or a derivative or        mimetic thereof or an amino acid sequence having at least about        45% similarity to at least 10 contiguous amino acids in SEQ ID        NO:2.

The protein of the present invention is preferably in isolated form. By“isolated” is meant a protein having undergone at least one purificationstep and this is conveniently defined, for example, by a compositioncomprising at least about 10% subject protein, preferably at least about20%, more preferably at least about 30%, still more preferably at leastabout 40-50%, even still more preferably at least about 60-70%, yet evenstill more preferably 80-90% or greater of subject protein relative toother components as determined by molecular weight, amino acid sequenceor other convenient means. The protein of the present invention may alsobe considered, in a preferred embodiment, to be biologically pure.

The sphingosine kinase of the present invention may be in multimericform meaning that two or more molecules are associated together. Wherethe same sphingosine kinase molecules are associated together, thecomplex is a homomultimer. An example of a homomultimer is a homodimer.Where at least one sphingosine kinase is associated with at least onenon-sphingosine kinase molecule, then the complex is a heteromultimersuch as a heterodimer.

The ability to produce recombinant sphingosine kinase permits the largescale production of sphingosine kinase for commercial use. Thesphingosine kinase may need to be produced as part of a large peptide,polypeptide or protein which may be used as is or may first need to beprocessed in order to remove the extraneous proteinaceous sequences.Such processing includes digestion with proteases, peptidases andamidases or a range of chemical, electrochemical, sonic or mechanicaldisruption techniques.

Notwithstanding that the present invention encompasses recombinantproteins, chemical synthetic techniques are also preferred in synthesisof sphingosine kinase.

Sphingosine kinase according to the present invention is convenientlysynthesized based on molecules isolated from the human. Isolation of thehuman molecules may be accomplished by any suitable means such as bychromotographic separation, for example using CM-cellulose ion exchangechromatography followed by Sephadex (e.g. G-50 column) filtration. Manyother techniques are available including HPLC, PAGE amongst others.

Sphingosine kinase may be synthesized by solid phase synthesis usingF-moc chemistry as described by Carpino et al. (1991). Sphingosinekinase and fragments thereof may also be synthesized by alternativechemistries including, but not limited to, t-Boc chemistry as describedin Stewart et al. (1985) or by classical methods of liquid phase peptidesynthesis.

Without limiting the theory or mode of action of the present invention,sphingosine kinase is a key regulatory enzyme in the activity of thesphingosine kinase signalling pathway. By “sphingosine kinase signallingpathway” is meant a signalling pathway which utilizes one or both ofsphingosine kinase and/or sphingosine-1-phosphate. It is thought that asphingosine kinase signalling pathway cascade which results in adhesionmolecule expression may take the form of:

-   -   (i) the generation of ceramide from sphingomyelin via S. Mase        activity, said ceramide being converted to sphingosine;    -   (ii) sphingosine-1-phosphate (referred to hereinafter as        “Sph-1-P”) generation by stimulation of sphingosine kinase; and    -   (iii) the activation of MEK/ERK and nuclear translocation of        NF-κB downstream from Sph-1-P generation.

The sphingosine kinase signaling pathway is known to regulate cellularactivities such as those which lead to inflammation, apoptosis and cellproliferation. For example, upregulation of the production ofinflammatory mediators such as cytokines, chemokines, eNOS andupregulation of adhesion molecule expression. Said upregulation may beinduced by a number of stimuli including, for example, inflammatorycytokines such as tumour necrosis factor-α (TNF-α) and interleukin-1(IL-1), endotoxin, oxidized or modified lipids, radiation or tissueinjury.

The cloning and sequencing of this gene and its expression product nowprovides additional molecules for use in the prophylactic andtherapeutic treatment of diseases characterised by unwanted cellularactivity, which activity is either directly or indirectly modulated viathe activity of the sphingosine kinase signaling pathway. Examples ofdiseases involving unwanted sphingosine kinase regulated cellularactivity include rheumatoid arthritis, asthma, atherosclerosis,meningitis, multiple sclerosis and septic shock. Accordingly, thepresent invention contemplates therapeutic and prophylactic uses ofsphingosine kinase amino acid and nucleic acid molecules, in addition tosphingosine kinase agonistic and antagonistic agents, for the regulationof cellular functional activity, such as for example, regulation ofinflammation.

The present invention contemplates, therefore, a method for modulatingexpression of sphingosine kinase in a subject, said method comprisingcontacting the sphingosine kinase gene with an effective amount of anagent for a time and under conditions sufficient to up-regulate ordown-regulate or otherwise modulate expression of sphingosine kinase.For example, sphingosine kinase antisense sequences such asoligonucleotides may be introduced into a cell to down-regulate one ormore specific functional activities of that cell. Conversely, a nucleicacid molecule encoding sphingosine kinase or a derivative thereof may beintroduced to up-regulate one or more specific functional activities ofany cell not expressing the endogenous sphingosine kinase gene.

Another aspect of the present invention contemplates a method ofmodulating activity of sphingosine kinase in a mammal, said methodcomprising administering to said mammal a modulating effective amount ofan agent for a time and under conditions sufficient to increase ordecrease sphingosine kinase activity.

Modulation of said activity by the administration of an agent to amammal can be achieved by one of several techniques, including but in noway limited to introducing into said mammal a proteinaceous ornon-proteinaceous molecule which:

-   -   (i) modulates expression of sphingosine kinase;    -   (ii) functions as an antagonist of sphingosine kinase;    -   (iii) functions as an agonist of sphingosine kinase.

Said proteinaceous molecule may be derived from natural or recombinantsources including fusion proteins or following, for example, naturalproduct screening. Said non-proteinaceous molecule may be, for example,a nucleic acid molecule or may be derived from natural sources, such asfor example natural product screening or may be chemically synthesized.The present invention contemplates chemical analogs of sphingosinekinase or small molecules capable of acting as agonists or antagonistsof sphingosine kinase. Chemical agonists may not necessarily be derivedfrom sphingosine kinase but may share certain conformationalsimilarities. Alternatively, chemical agonists may be specificallydesigned to mimic certain physiochemical properties of sphingosinekinase. Antagonists may be any compound capable of blocking, inhibitingor otherwise preventing sphingosine kinase from carrying out its normalbiological functions. Antagonists include monoclonal antibodies specificfor sphingosine kinase, or parts of sphingosine kinase, and antisensenucleic acids which prevent transcription or translation of sphingosinekinase genes or mRNA in mammalian cells. Modulation of sphingosinekinase expression may also be achieved utilizing antigens, RNA,ribosomes, DNAzymes, RNA aptamers or antibodies.

Said proteinaceous or non-proteinaceous molecule may act either directlyor indirectly to modulate the expression of sphingosine kinase or theactivity of sphingosine kinase. Said molecule acts directly if itassociates with sphingosine kinase or sphingosine kinase to modulate theexpression or activity of sphingosine kinase or sphingosine kinase. Saidmolecule acts indirectly if it associates with a molecule other thansphingosine kinase or sphingosine kinase which other molecule eitherdirectly or indirectly modulates the expression or activity ofsphingosine kinase or sphingosine kinase. Accordingly, the method of thepresent invention encompasses the regulation of sphingosine kinase orsphingosine kinase expression or activity via the induction of a cascadeof regulatory steps which lead to the regulation of sphingosine kinaseor sphingosine kinase expression or activity.

Another aspect of the present invention contemplates a method ofmodulating cellular functional activity in a mammal said methodcomprising administering to said mammal an effective amount of an agentfor a time and under conditions sufficient to modulate the expression ofa nucleotide sequence encoding sphingosine kinase or sufficient tomodulate the activity of sphingosine kinase.

Yet another aspect of the present invention contemplates a method ofmodulating cellular functional activity in a mammal said methodcomprising administering to said mammal an effective amount ofsphingosine kinase or sphingosine kinase.

The sphingosine kinase, sphingosine kinase or agent used may also belinked to a targeting means such as a monoclonal antibody, whichprovides specific delivery of the sphingosine kinase, sphingosine kinaseor agent to the target cells.

In a preferred embodiment of the present invention, the sphingosinekinase, sphingosine kinase or agent used in the method is linked to anantibody specific for said target cells to enable specific delivery tothese cells.

Reference to “modulating cellular functional activity” is a reference toup-regulating, down-regulating or otherwise altering any one or more ofthe activities which a cell is capable of performing such as, but notlimited to, one or more of chemokine production, cytokine production,nitric oxide synthesase, adhesion molecule expression and production ofother inflammatory modulators.

Administration of the sphingosine kinase, sphingosine kinase or agent,in the form of a pharmaceutical composition, may be performed by anyconvenient means. Sphingosine kinase, sphingosine kinase or agent of thepharmaceutical composition are contemplated to exhibit therapeuticactivity when administered in an amount which depends on the particularcase. The variation depends, for example, on the human or animal and thesphingosine kinase, sphingosine kinase or agent chosen. A broad range ofdoses may be applicable. Considering a patient, for example, from about0.1 mg to about 1 mg of sphingosine kinase or agent may be administeredper kilogram of body weight per day. Dosage regimes may be adjusted toprovide the optimum therapeutic response. For example, several divideddoses may be administered daily, weekly, monthly or other suitable timeintervals or the dose may be proportionally reduced as indicated by theexigencies of the situation. The sphingosine kinase or agent may beadministered in a convenient manner such as by the oral, intravenous(where water soluble), intranasal, intraperitoneal, intramuscular,subcutaneous, intradermal or suppository routes or implanting (e.g.using slow release molecules). With particular reference to use ofsphingosine kinase or agent, these peptides may be administered in theform of pharmaceutically acceptable nontoxic salts, such as acidaddition salts or metal complexes, e.g. with zinc, iron or the like(which are considered as salts for purposes of this application).Illustrative of such acid addition salts are hydrochloride,hydrobromide, sulphate, phosphate, maleate, acetate, citrate, benzoate,succinate, malate, ascorbate, tartrate and the like. If the activeingredient is to be administered in tablet form, the tablet may containa binder such as tragacanth, corn starch or gelatin; a disintegratingagent, such as alginic acid; and a lubricant, such as magnesiumstearate.

A further aspect of the present invention relates to the use of theinvention in relation to mammalian disease conditions. For example, thepresent invention is particularly useful, but in no way limited to, usein inflammatory diseases.

Accordingly, another aspect of the present invention relates to a methodof treating a mammal said method comprising administering to said mammalan effective amount of an agent for a time and under conditionssufficient to modulate the expression of sphingosine kinase orsufficient to modulate the activity of sphingosine kinase wherein saidmodulation results in modulation of cellular functional activity.

In another aspect the present invention relates to a method of treatinga mammal said method comprising administering to said mammal aneffective amount of sphingosine kinase or sphingosine kinase for a timeand under conditions sufficient to modulate cellular functionalactivity.

Yet another aspect of the present invention relates to the use of anagent capable of modulating the expression of sphingosine kinase ormodulating the activity of sphingosine kinase in the manufacture of amedicament for the modulation of cellular functional activity.

A further aspect of the present invention relates to the use ofsphingosine kinase or sphingosine kinase in the manufacture of amedicament for the modulation of cellular functional activity.

Still yet another aspect of the present invention relates to agents foruse in modulating sphingosine kinase expression or sphingosine kinaseactivity wherein said modulation results in modulation of cellularfunctional activity.

Another aspect of the present invention relates to sphingosine kinase orsphingosine kinase for use in modulating cellular functional activity.

In a related aspect of the present invention, the mammal undergoingtreatment may be a human or an animal in need of therapeutic orprophylactic treatment.

In yet another further aspect the present invention contemplates apharmaceutical composition comprising sphingosine kinase, sphingosinekinase or an agent capable of modulating sphingosine kinase expressionor sphingosine kinase activity together with one or morepharmaceutically acceptable carriers and/or diluents. Sphingosinekinase, sphingosine kinase or said agent are referred to as the activeingredients.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating such as licithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsuperfactants. The preventions of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredient into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof.

When sphingosine kinase, sphingosine kinase and sphingosine kinasemodulators are suitably protected they may be orally administered, forexample, with an inert diluent or with an assimilable edible carrier, orthey may be enclosed in hard or soft shell gelatin capsule, or they maybe compressed into tablets, or they may be incorporated directly withthe food of the diet. For oral therapeutic administration, the activecompound may be incorporated with excipients and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 1% by weight of active compound.The percentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 5 to about 80% of theweight of the unit. The amount of active compound in suchtherapeutically useful compositions in such that a suitable dosage willbe obtained. Preferred compositions or preparations according to thepresent invention are prepared so that an oral dosage unit form containsbetween about 0.1 μg and 2000 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain thefollowing: A binder such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such asucrose, lactose or saccharin may be added or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier. Various other materials may be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills, or capsules may be coated with shellac,sugar or both. A syrup or elixir may contain the active compound,sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring such as cherry or orange flavor. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compound may be incorporated intosustained-release preparations and formulations.

Pharmaceutically acceptable carriers and/or diluents include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, use thereof in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the novel dosageunit forms of the invention are dictated by and directly dependent on(a) the unique characteristics of the active material and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active material for the treatment ofdisease in living subjects having a diseased condition in which bodilyhealth is impaired.

The principal active ingredient is compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically acceptable carrier in dosage unit form as hereinbeforedisclosed. A unit dosage form can, for example, contain the principalactive compound in amounts ranging from 0.5 μg to about 2000 mg.Expressed in proportions, the active compound is generally present infrom about 0.5 μg to about 2000 mg/ml of carrier. In the case ofcompositions containing supplementary active ingredients, the dosagesare determined by reference to the usual dose and manner ofadministration of the said ingredients.

The pharmaceutical composition may also comprise genetic molecules suchas a vector capable of transfecting target cells where the vectorcarries a nucleic acid molecule capable of expressing sphingosinekinase, modulating sphingosine kinase expression or sphingosine kinaseactivity. The vector may, for example, be a viral vector.

Sphingosine kinase can also be utilized to create gene knockout modelsin either cells or animals, which knocked out gene is the sphingosinekinase gene expressed by said cells or animals. Accordingly in anotheraspect the present invention should be understood to extend to methodsof creating sphingosine kinase gene cell or animal knockout modelswherein sphingosine kinase has been utilized to facilitate knocking outof the endogenous sphingosine kinase gene of said cell or animal, and tothe knockout models produced therefrom.

Still another aspect of the present invention is directed to antibodiesto sphingosine kinase including catalytic antibodies. Such antibodiesmay be monoclonal or polyclonal and may be selected from naturallyoccurring antibodies to sphingosine kinase or may be specifically raisedto sphingosine kinase. In the case of the latter, sphingosine kinase mayfirst need to be associated with a carrier molecule. The antibodiesand/or recombinant sphingosine kinase of the present invention areparticularly useful as therapeutic or diagnostic agents. Alternatively,fragments of antibodies may be used such as Fab fragments. Furthermore,the present invention extends to recombinant and synthetic antibodiesand to antibody hybrids. A “synthetic antibody” is considered herein toinclude fragments and hybrids of antibodies. The antibodies of thisaspect of the present invention are particularly useful forimmunotherapy and may also be used as a diagnostic tool, for example,for monitoring the program of a therapeutic regime.

For example, sphingosine kinase can be used to screen for naturallyoccurring antibodies to sphingosine kinase. These may occur, for examplein some inflammatory disorders.

For example, specific antibodies can be used to screen for sphingosinekinase proteins. The latter would be important, for example, as a meansfor screening for levels of sphingosine kinase in a cell extract orother biological fluid or purifying sphingosine kinase made byrecombinant means from culture supernatant fluid. Techniques for theassays contemplated herein are known in the art and include, forexample, sandwich assays, ELISA and flow cytometry.

It is within the scope of this invention to include any secondantibodies (monoclonal, polyclonal or fragments of antibodies) directedto the first mentioned antibodies discussed above. Both the first andsecond antibodies may be used in detection assays or a first antibodymay be used with a commercially available anti-immunoglobulin antibody.An antibody as contemplated herein includes any antibody specific to anyregion of sphingosine kinase.

Both polyclonal and monoclonal antibodies are obtainable by immunizationwith the protein or peptide derivatives and either type is utilizablefor immunoassays. The methods of obtaining both types of sera are wellknown in the art. Polyclonal sera are less preferred but are relativelyeasily prepared by injection of a suitable laboratory animal with aneffective amount of sphingosine kinase, or antigenic parts thereof,collecting serum from the animal, and isolating specific sera by any ofthe known immunoadsorbent techniques. Although antibodies produced bythis method are utilizable in virtually any type of immunoassay, theyare generally less favored because of the potential heterogeneity of theproduct.

The use of monoclonal antibodies in an immunoassay is particularlypreferred because of the ability to produce them in large quantities andthe homogeneity of the product. The preparation of hybridoma cell linesfor monoclonal antibody production derived by fusing an immortal cellline and lymphocytes sensitized against the immunogenic preparation canbe done by techniques which are well known to those who are skilled inthe art. (See, for example Douillard and Hoffman, Basic Facts aboutHybridomas, in Compendium of Immunology Vol. II, ed. by Schwartz, 1981;Kohler and Milstein, Nature 256: 495-499, 1975; European Journal ofImmunology 6: 511-519, 1976).

In another aspect of the present invention, the molecules of the presentinvention are also useful as screening targets for use in applicationssuch as the diagnosis of disorders which are regulated by sphingosinekinase.

Yet another aspect of the present invention contemplates a method fordetecting sphingosine kinase or sphingosine kinase mRNA in a biologicalsample from a subject said method comprising contacting said biologicalsample with an antibody specific for sphingosine kinase or sphingosinekinase mRNA or its derivatives or homologs for a time and underconditions sufficient for an antibody-sphingosine kinase orantibody-sphingosine kinase mRNA complex to form, and then detectingsaid complex.

The presence of sphingosine kinase may be determined in a number of wayssuch as by Western blotting, ELISA or flow cytometry procedures.Sphingosine kinase mRNA may be detected, for example, by in situhybridization or Northern blotting. These, of course, include bothsingle-site and two-site or “sandwich” assays of the non-competitivetypes, as well as in the traditional competitive binding assays. Theseassays also include direct binding of a labeled antibody to a target.

Sandwich assays are among the most useful and commonly used assays andare favored for use in the present invention. A number of variations ofthe sandwich assay technique exist, and all are intended to beencompassed by the present invention. Briefly, in a typical forwardassay, an unlabelled antibody is immobilized on a solid substrate andthe sample to be tested brought into contact with the bound molecule.After a suitable period of incubation, for a period of time sufficientto allow formation of an antibody-antigen complex, a second antibodyspecific to the antigen, labeled with a reporter molecule capable ofproducing a detectable signal is then added and incubated, allowing timesufficient for the formation of another complex ofantibody-antigen-labeled antibody. Any unreacted material is washedaway, and the presence of the antigen is determined by observation of asignal produced by the reporter molecule. The results may either bequalitative, by simple observation of the visible signal, or may bequantitated by comparing with a control sample containing known amountsof hapten. Variations on the forward assay include a simultaneous assay,in which both sample and labeled antibody are added simultaneously tothe bound antibody. These techniques are well known to those skilled inthe art, including any minor variations as will be readily apparent. Inaccordance with the present invention the sample is one which mightcontain sphingosine kinase including cell extract, tissue biopsy orpossibly serum, saliva, mucosal secretions, lymph, tissue fluid andrespiratory fluid. The sample is, therefore, generally a biologicalsample comprising biological fluid but also extends to fermentationfluid and supernatant fluid such as from a cell culture.

In the typical forward sandwich assay, a first antibody havingspecificity for the sphingosine kinase or antigenic parts thereof, iseither covalently or passively bound to a solid surface. The solidsurface is typically glass or a polymer, the most commonly used polymersbeing cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chlorideor polypropylene. The solid supports may be in the form of tubes, beads,discs of microplates, or any other surface suitable for conducting animmunoassay. The binding processes are well-known in the art andgenerally consist of cross-linking covalently binding or physicallyadsorbing, the polymer-antibody complex is washed in preparation for thetest sample. An aliquot of the sample to be tested is then added to thesolid phase complex and incubated for a period of time sufficient (e.g.2-40 minutes) and under suitable conditions (e.g. 25° C.) to allowbinding of any subunit present in the antibody. Following the incubationperiod, the antibody subunit solid phase is washed and dried andincubated with a second antibody specific for a portion of the hapten.The second antibody is linked to a reporter molecule which is used toindicate the binding of the second antibody to the hapten.

An alternative method involves immobilizing the target molecules in thebiological sample and then exposing the immobilized target to specificantibody which may or may not be labeled with a reporter molecule.Depending on the amount of target and the strength of the reportermolecule signal, a bound target may be detectable by direct labelingwith the antibody. Alternatively, a second labeled antibody, specific tothe first antibody is exposed to the target-first antibody complex toform a target-first antibody-second antibody tertiary complex. Thecomplex is detected by the signal emitted by the reporter molecule.

By “reporter molecule” as used in the present specification, is meant amolecule which, by its chemical nature, provides an analyticallyidentifiable signal which allows the detection of antigen-boundantibody. Detection may be either qualitative or quantitative. The mostcommonly used reporter molecules in this type of assay are eitherenzymes, fluorophores or radionuclide containing molecules (i.e.radioisotopes) and chemiluminescent molecules.

In the case of an enzyme immunoassay, an enzyme is conjugated to thesecond antibody, generally by means of glutaraldehyde or periodate. Aswill be readily recognized, however, a wide variety of differentconjugation techniques exist, which are readily available to the skilledartisan. Commonly used enzymes include horseradish peroxidase, glucoseoxidase, beta-galactosidase and alkaline phosphatase, amongst others.The substrates to be used with the specific enzymes are generally chosenfor the production, upon hydrolysis by the corresponding enzyme, of adetectable color change. Examples of suitable enzymes include alkalinephosphatase and peroxidase. It is also possible to employ fluorogenicsubstrates, which yield a fluorescent product rather than thechromogenic substrates noted above. In all cases, the enzyme-labeledantibody is added to the first antibody hapten complex, allowed to bind,and then the excess reagent is washed away. A solution containing theappropriate substrate is then added to the complex ofantibody-antigen-antibody. The substrate will react with the enzymelinked to the second antibody, giving a qualitative visual signal, whichmay be further quantitated, usually spectrophotometrically, to give anindication of the amount of hapten which was present in the sample.“Reporter molecule” also extends to use of cell agglutination orinhibition of agglutination such as red blood cells on latex beads, andthe like.

Alternately, fluorescent compounds, such as fluorecein and rhodamine,may be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labeled antibody adsorbs the light energy,inducing a state to excitability in the molecule, followed by emissionof the light at a characteristic color visually detectable with a lightmicroscope. As in the EIA, the fluorescent labeled antibody is allowedto bind to the first antibody-hapten complex. After washing off theunbound reagent, the remaining tertiary complex is then exposed to thelight of the appropriate wavelength the fluorescence observed indicatesthe presence of the hapten of interest. Immunofluorescene and EIAtechniques are both very well established in the art and areparticularly preferred for the present method. However, other reportermolecules, such as radioisotope, chemiluminescent or bioluminescentmolecules, may also be employed.

The present invention also contemplates genetic assays such as involvingPCR analysis to detect sphingosine kinase or its derivatives.

Further features of the present invention are more fully described inthe following non-limiting examples.

EXAMPLE 1 Purification and Cloning of Human SphingosineKinase—Experimental Procedures

Materials

D-erythro-Sphingosine, D-erythro-dihydrosphingosine,DL-threo-dihydrosphingosine, N,N-dimethylsphingosine,N-acetylsphingosine (C₂-ceramide), S1P and Fumosin B1 were purchasedfrom Biomol Research Laboratories Inc. (Plymouth Meeting, Pa.).Phytosphingosine, L-α-phosphatidic acid, L-α-phosphatidylinositol,L-α-phosphatidylserine, L-α-phosphatidylcholine,L-α-phosphatidylethanolamine, 1,2-dioctanoyl-sn-glycerol,1,2-dioleoyl-sn-glycerol, ATP, calmodulin, glutathione and bovine serumalbumin (BSA) were from Sigma. N,N,N-trimethylsphingosine and ADP werepurchased from Calbiochem (Band Soden, Germany), [γ³²P]ATP fromGeneworks (Adelaide, South Australia), TNFα from R&D Systems Inc.(Minneapolis, Minn.), and isopropyl-β-D-thiogalactoside (IPTG) fromPromega (Madison, Wis.). Prepacked Mono-Q, Superose 75 and HiTrap-Qcolumns, Q Sepharose fast flow, calmodulin Sepharose 4B, glutathioneSepharose 4B, thrombin and gel filtration molecular mass proteinstandards were from Amersham Pharmacia Biotech. SDS-PAGE molecular massprotein standards, silver stain plus kit, Coomassie Brilliant Blue R250and Coomassie protein reagent were from Bio-Rad. Centricon concentratorswere purchased from Amicon Inc. (Beverly, Mass.) and BCA protein reagentwas from Pierce Chemical Company (Rockford, Ill.).

Sphingosine Kinase Enzyme Assay

Sphingosine kinase activity was routinely determined usingD-erythro-sphingosine and [γ³²P]ATP as substrates, essentially aspreviously described (Olivera et al., 1998) with some modifications.Briefly, assays were performed by incubating samples at 37° C. for 30min with sphingosine (100 μM stock dissolved in 5% Triton X-100) and[γ-³²P]ATP (1 mM; 10 μCi/ml) in assay buffer containing 100 mM Tris/HCl(pH 7.4), 10 mM MgCl₂ 10% (v/v) glycerol, 1 mM dithiothreitol, 1 mMEGTA, 1 mM Na₃VO₄, 15 mM NaF, 0.5 mM 4-deoxypyridoxine in a total volumeof 100 μl. Reactions were terminated and sphingosine-1-phosphateextracted by the addition of 700 μl of chloroform/methanol/HCl(100:200:1, v/v), followed by vigorous mixing, addition of 200 μl ofchloroform and 200 μl of 2 M KCl, and phase separation bycentrifugation. The labeled S1P in the organic phase was isolated by TLCon Silica Gel 60 with 1-butanol/ethanol/acetic acid/water (8:2:1:2, v/v)and quantitated by phosphorimager (Molecular Dynamics, Sunnyvale,Calif.). One unit (U) of activity is defined as 1 pmol of S1P formed perminute.

Purification of Sphingosine Kinase from Human Placenta

Sphingosine kinase was purified from 1240 g of human placenta (4placentas), with all steps performed at 4° C. The placentas were diced,washed in buffer A (25 mM Tris/HCl buffer, pH 7.4 containing 10% (v/v)glycerol, 0.05% Triton X-100 and 1 mM dithiothreitol), transfered to 1.5L of fresh buffer A containing a protease inhibitor cocktail (Complete™;Boehringer Mannheim) (buffer B), and minced in a Waring blender. Theresultant homogenate was stored on ice for 30 min to enhance enzymeextraction, and the soluble fraction of the homogenate then isolated bycentrifugation at 17 000 g for 60 min. This preparation was thenfractionated by (NH₄)₂SO₄ precipitation by the addition of solid(NH₄)₂SO₄ at pH 7.4 and collection of the precipitated proteins bycentrifugation (17 000 g, 30 min). The 25-35%-saturated (NH₄)₂SO₄fraction was then redissolved in a buffer B, desalted by extensivedialysis against this same buffer, and centrifuged (17 000 g, 30 min) toremove insoluble material. All subsequent chomatographic steps wereperformed using a FPLC system (Pharmacia Biotech) at 4° C.

The dialyzed (NH₄)₂SO₄ fraction was applied to a Q-Sepharose fast flowcolumn (50 mm diameter, 250 ml bed volume) pre-equilibrated with bufferA at a flow rate of 7 ml/min. Sphingosine kinase activity was elutedwith a NaCl gradient of 0 to 1M in buffer A and collected in 10 mlfractions. Fractions containing highest SK1 activity were then combined,and CaCl₂ and NaCl added to give final concentrations of 4 mM and 250mM, respectively. This pooled extract was then applied to acalmodulin-Sepharose 4B column (16 mm diameter, 10 ml bed volume),pre-equilibrated with buffer A containing 2 mM CaCl₂, at a flow rate of1 ml/min. The column was then washed with several column volumes ofequilibration buffer, followed by buffer A containing 4 mM EGTA, andthen SKI eluted with buffer A containing 4 mM EGTA and 1 M NaCl. Thefractions containing highest sphingosine kinase activity were pooled,desalted on a Sephadex G-25 column, and applied at a flow rate of 1ml/min to a Mono-Q column (5 mm diameter, 1 ml bed volume)pre-equilibrated with buffer A. Sphingosine kinase activity was elutedwith a NaCl gradient to 0 to 1M in buffer A. NaCl (to 500 mM) wasimmediately added to the fractions (1 ml) collected to stabilize enzymeactivity. The Mono-Q fractions containing highest sphingosine kinaseactivity were combined and desalted on a Sephadex G-25 column. ATP andMgCl₂ were then added to the pooled fractions to a final concentrationsof 1 mM and 5 mM, respectively, before reapplication at a flow rate of 1ml/min to the Mono-Q column pre-equilibrated with buffer A containing 1mM ATP and 5 mM MgCl₂. Sphingosine kinase activity was eluted with aNaCl gradient of 0 to 1M in the equilibration buffer. Again, NaCl (to500 mM) was immediately added to the fractions (1 ml) collected tostabilize enzyme activity. The ATP-Mono-Q fractions containing highestsphingosine kinase activity were pooled and concentrated 10-fold to afinal volume of 200 μl in a Centricon-10 concentrator and applied at aflow rate of 0.4 ml/min to a Superdex 75 column (10 mm diameter, 20 mlbed volume) pre-equilibrated with buffer A containing 500 mM NaCl.Sphingosine kinase activity was eluted with the same buffer and 0.4 mlfractions collected. The molecular mass of the enzyme was estimated fromthis column by comparison to the elution volumes of ribonuclease A,chymotrypsinogen A, ovalbumin and BSA.

Cloning of Human Sphingosine Kinase

The human sphingosine kinase (hSK) was amplified from a HUVEC λ Zap cDNAlibrary using PCR primers derived from human EST sequences (GenBank™accession numbers D31133, W63556, AA026479, AA232791, AA081152, AI769914and AI769914) aligned to the murine sphingosine kinases (Olivera et al.,1998). These primers, spanning a central SacII site (P1,5′-CGGAATTCCCAGTCGGCCGCGGTA-3′ [SEQ ID NO:3] and P2,5′-TAGAATTCTACCGCGGCCGACTGGCT-3′ [SEQ ID NO:4]), were used incombination with T3 and T7 primers to generate two overlapping PCRproducts of 669 bp and 550 bp that represented the 5′ and 3′ ends ofhSK, respectively. These two PCR products were then separately clonedinto pGEM4Z. A 584 bp SacII fragment from the 5′ hSK PCR clone was thensub-cloned in the correct orientation into the SacII site of the 3′ hSKPCR clone, to generate a 1130 bp partial hSK cDNA clone. A full lengthclone encoding hSK was then generated by sub-cloning a 120 bp EcoRI/StuIfragment from the 669 bp 5′ hSK clone into this pGEM4Z-1130 bp clonedigested with EcoRI/StuI. Sequencing the cDNA clone in both directionsverified the integrity of the hSK cDNA sequence.

For mammalian cell expression the hSK cDNA was FLAG epitope tagged atthe 3′-end by PFU polymerase PCR with oligonucleotide primers T7 and5′-TAGAATTCACTTGTCATCGTCGTCCTTGTAGTCTAAGGGCTCTTCTGGCGGT-3′ [SEQ IDNO:5]. This FLAG-tagged hSK cDNA was then cloned into pcDNA3 bydigestion with EcoRI. The orientation was determined by restrictionanalysis and sequencing verified the integrity of the hSK-FLAG cDNAsequence. For bacterial expression, the full length hSK cDNA wassub-cloned into pGEX4T2. The pGEM4Z-hSK clone was digested with BamHIand blunted with 3U PFU polymerase in 1×PFU buffer, 50 μM dNTP's at 72°C. for 30 minutes. The 1163 bp hSK cDNA was gel purified followingdigestion with SalI and the blunt/SalI fragment was then ligated topGEX4T2 SmaI/XhoI.

Sphingosine Kinase Amino Acid Sequence Analysis

The human sphingosine kinase amino acid sequence was searched againstnon-redundant amino acid and nucleotide sequence databases at theAustralian National Genome Information Service using the blastp andtblastn algorithms (Altschul et al., 1990).

Cell Culture

Human umbilical vein endothelial cells (HUVEC) were isolated aspreviously described (Wall et al., 1978) and cultured on gelatin-coatedculture flasks in medium M199 with Earle's salts supplemented with 20%fetal calf serum, 25 μg/ml endothelial growth supplement (CollaborativeResearch) and 25 μg/ml heparin. The cells were passaged three times andgrown to 80% confluency before treatment and harvesting. Human embryonickidney cells (HEK293, ATCC CRL-1573) cells were cultured on Dulbecco'smodified Eagle's medium containing 10% fetal calf serum, 2 mM glutamine,0.2% (w/v) sodium bicarbonate, penicillin (1.2 mg/ml), and gentamycin(1.6 mg/ml). HEK293 cells were transiently transfected using the calciumphosphate precipitation method (Graham & van der Eb, 1973). Treatment ofHUVEC and HEK293 cells with TNFα (1 ng/ml) was performed as previouslydescribed (Xia et al., 1998).

EXAMPLE 2 Expression and Isolation of Recombinant Human SphingosineKinase from E. Coli—Experimental Procedure

The full length SPHK cDNA cloned into pGEX4T2 was transformed into E.coli BL21. Overnight cultures (100 ml) of transformed isolates weregrown with shaking (200 rpm) at 30° C. in Superbroth (20 g/L glucose, 35g/L tryptone, 20 g/L yeast extract, 5 g/L NaCl, pH 7.5) mediumcontaining ampicillin (100 mg/L). The cultures were diluted 1:20 infresh medium and grown at 30° C. with shaking to an OD₆₀₀ of 0.6-0.7.Expression of the glutathione-s-transferase (GST)-coupled sphingosinekinase (GST-SK) was then induced by addition of 0.1 mMisopropyl-β-D-thiogalactoside and further incubation of the cultures at30° C. for 3 h. After this time the bacterial cells were then harvestedby centrifugation at 6,000 g for 20 min at 4° C. and resuspended in 20ml of buffer B containing 250 mM NaCl. The cells were then lysed withlysozyme at a final concentration of 0.3 mg/ml for 15 min at 25° C.followed by sonication, consisting of three cycles of 20 s ultrasonicpulses followed by one minute cooling. The lysate was then clarified bycentrifugation at 50,000 g for 45 min at 4° C., followed by filtratingthrough 0.22 μm filters. To be filtered supernatant was then incubatedwith 0.2 volumes of 50% (w/v) glutathione-Sepharose 4B (Pharmacia) thatwas washed and pro-equilibrated with buffer B, for 60 min at 4° C. withconstant mixing. After this time the mixture was poured into a glasschromatography column (10 mm diam.) and the beads (with bound GST-SK)washed with 10 column volumes of buffer B at 4° C. The GST-SK was theneluted from the column in 10 ml of buffer B containing 10 mM reducedglutathione. Cleavage of the GST away from sphingosine kinase was thenperformed by incubation with 20 μg (30 N.I.H. units) thrombin(Pharmacia) for 3 h at 25° C. The released sphingosine kinase was thenpurified by application of the cleavage mix to a calmodulin-Sepharosecolumn and then a Mono-Q anion exchange column for the purification ofthe sphingosine kinase from human placenta. These columns resulted inpurification of the recombinant sphingosine kinase to homogeneity.

EXAMPLE 3 Characterization of Sphingosine Kinases—ExperimentalProcedures

The effect of pH on the activity of the isolated sphingosine kinases wasdetermined over the pH range 4.0 to 11.0 in 50 mM buffers (sodiumacetate, pH 4.0-5.0; Mes, pH 6.0-7.0; Hepes, pH 7.0-8.2; Tris/HCl, pH8.2-10.0; Caps, pH 10.0-11.0) at 37° C. pH stability was determined byassaying the residual activity after pre-incubation of the enzymes inthe same buffers for 5 h at 4° C. Similarly, thermal stabilities weredetermined by assaying the residual activity after pre-incubation of theenzymes at various temperatures (4-80° C.) for 30 min at pH 7.4 (50 mMTris/HCl containing 10% glycerol, 0.5 M NaCl and 0.05% Triton X-100).Substrate kinetics were analyzed using Michaelis-Menten kinetics with aweighted non-linear regression program (Easterby, 1996). Sincesphingosine and its analogues were added to the enzyme assays in mixedmicelles with Triton X-100, where they exhibit surface dilution kinetics(Buehrer and Bell, 1992), all K_(m) and K_(i) values obtained for thesemolecules were expressed as mol % of Triton X-100, rather than as bulksolution concentrations. For assays to determine the effect ofcalcium/calmodulin on sphingosine kinase activity, calcium andcalmodulin were added to the standard assay mixtures containing 20 nM ofisolated sphingosine kinase at final concentrations of 4 mM and 0.6 μM,respectively.

EXAMPLE 4 Other Analytical Methods

Protein was determined using either the Coomassie Brilliant Blue(Bradford et al., 1976) or Bicinchoninic acid (Smith et al., 1985)reagents using BSA as standard. In some cases protein estimations wereperformed after concentration and removal of detergent by precipitation(Wessel and Flügge, 1984) to increase the sensitivity and accuracy ofthe determinations. SDS-PAGE was performed according to the method ofLaemmli (1970) using 12% acrylamide gels. Protein bands on gels werevisualized with either Coomassie Brilliant Blue R250 or silver staining.Molecular mass was estimated by comparison to the electophoriticmobility of myosin, β-galactosidase, BSA, ovalbuminm, carbonicanhydrase, soybean trypsin inhibitor, lysozyme and aprotinin.

EXAMPLE 5 Purification of Human Sphingosine Kinase—Results

Just over half of the total sphingosine kinase activity in humanplacenta was present in the cytosol after tissue homogenization (Table3). The purification of sphingosine kinase from human placenta issummarized in Table 4. The soluble fraction from the homogenate of fourhuman placentae was initially subjected to ammonium sulphateprecipitation. This resulted in a remarkably good purification ofsphingosine kinase (33-fold), which precipitated in the 25-35% saturatedammonium sulphate fraction. For anion exchange chromatography theammonium sulphate fraction was rapidly desalted through the use ofSephadex G25 column and the desalted fraction loaded immediately onto aQ Sepharose FF column. The speed of this desalting step appearedcritical since the sphingosine kinase activity appeared very unstable atNaCl concentrations below about 0.2 M, meaning slower desalting steps,such as dialysis, resulted in substantial losses of enzyme activity.Application of a NaCl gradient of 0 to 1 M to the Q Sepharose FF columnresulted in two peaks of sphingosine kinase activity, eluting atapproximately 0.15 M and 0.6 M NaCl, and designated SK1 and SK2,respectively (FIG. 2). For this study SK1 was selected for furtherpurification due to its greater abundance and stability; SK2 activityappeared very unstable and, unlike SK1, could not be stabilized by theaddition of 0.5M NaCl, 10% glycerol and 0.05% Triton X-100. SKb 1 wasalso chosen since it appeared to be the isoform present in HUVEC, asdiscussed later.

Fractions from Q Sepharose FF column containing SK1 were then affinitypurified by application to a calmodulin-Sepharose 4B column in thepresence of 4 mM CaCl₂, and elution of SK1 performed with EGTA and NaCl,resulting in further substantial purification (38-fold) and high enzymeyields. SK1 could not be eluted from the calmodulin Sepharose 4B columnwith EGTA alone, indicating an unusual association of the enzyme withthis affinity matrix. The active fractions that eluted from thecalmodulin Sepharose 4B column were then desalted and applied to twosubsequent steps of analystical anion exchange chromatography on a MonoQ column, with the second step performed in the presence of 1 mM ATP and5 mM MgCl₂ (FIG. 3). The active fractions resulting from these anionexchange steps were then applied to gel filtration chromatography with aSuperdex 75 column as a final purification step. SK1 eluted from thiscolumn as a single peak with a molecule mass corresponding to 44 kDa.Analysis of the active fraction from this final column by SDS-PAGE withsilver staining (FIG. 4) revealed a single band of molecular mass 45kDa, indicating a homogenous protein that has been purified over amillion-fold from the original placenta extract with remarkably goodyield of 7% of the original sphingosine kinase activity (Table 4). Thisis the first sphingosine kinase to be purified to homogeneity from ahuman source.

EXAMPLE 6 Sphingosine Kinase Isoforms in HUVEC—Results

Since two sphingosine kinase activities were identified in humanplacenta (FIG. 2) we examined the multiplicity of this enzyme activityin HUVEC by the use of preparative anion exchange columns. In contrastto other human tissues and cells, application of HUVEC extracts to thesecolumns resulted in the appearance of only a single sphingosine kinasepeak that eluted at the same point as the human placenta SK1 (FIG. 5).Similarly, only a single sphingosine kinase peak eluted afterapplication of HUVEC extracts in which sphingosine kinase activity hadbeen stimulated by 10 min treatment of HUVEC with TNFα (Xia et al.,1999). These results would indicate that SK1, the human placentasphingosine kinase isolated in this study, is probably the main isoformfound in HUVEC, and that TNFA treatment results in an increase in theactivity of this enzyme, rather than the activation of another,otherwise latent, isoform.

EXAMPLE 7 Cloning of Human Sphingosine Kinase and Transient Exprsesionin HEK293 Cells—Results

A human sphingosine kinase cDNA was generated from a HUVEC λ Zap libraryusing primers designed from human ESTs aligned with the published murinesphingosine kinase sequence (Kohama et al., 1998). The cloning strategyis shown in FIG. 6 (prov). The cDNA has an apparent open reading framecoding for 384 amino acids (FIG. 7). It should be noted that thesequence lacked a recognizable Kozak consensus motif raising thepossibility that the actual initiation sequence may not be included inthis cDNA. The sphingosine kinase cDNA encodes for a protein (hSK) witha predicted isoelectric point of 6.64 and a molecular mass of 42,550kDa, consistent with the molecular mass determined for the purifiedhuman placenta sphingosine kinase (SK1). Subcloning into pcDNA3 andtransient expression of hSK in HEK293 cells resulted in a 3200-foldincrease in sphingosine kinase activity in these cells (FIG. 8),compared with untransfected HEK293 cells or HEK293 cells transfectedwith empty vector, indicating that the generated hSK cDNA encodes agenuine sphingosine kinase. Interestingly, although hSK-transfectedHEK293 cells had 3200-fold higher levels of sphingosine kinase activity,treatment of these cells with TNFα resulted in a rapid (10 min) increasein sphingosine kinase activity by a similar proportion (approximately2-fold) to that seen in untransfected HEK293 cells (FIG. 8) (Xia et al.,1998). This indicates the high levels of over-expressed sphingosinekinase are not saturating the TNFα mediated activation mechanism inthese cells.

EXAMPLE 8 Human Sphingosine Kinase Analysis—Results

A search of the database shown hSK has a high amino acid sequencesimilarity (28 to 36% identity) to two recently identified Saccharomycescerevisiae sphingosine kinases (Nagiec et al., 1998) and several otherESTs encoding putative sphingosine kinase proteins fromSchizosaccharomyces pombe, Caenorhabditis elegans and Arabidopisthaliana. Multiple sequence alignment of hSK with these homologues (FIG.9) revealed several regions of highly conserved amino acids through theprotein, but particularly towards the N-terminus.

A search of the domain structures of hSK sequence revealed threecalcium/calmodulin binding motifs (Rhoads & Friedberg, 1997), one of the1-8 14 type A ([FILVW]xxx{FAILVW]xx[FAILVW]xxxxx{FILVW] (SEQ ID NO:6)with net charge of +3 to +6) spanning residues 290 to 303, and two ofthe 1-8 14 type B ([FILVW]xxxxx{FAILVW]xxxxx[FILVW] (SEQ ID NO:7) withnet charge of +2 to +4) that overlap between residues 134 to 153.Further analysis of the hSK sequence revealed a possibleN-myristoylation site close to the N-terminus (at Gly⁵) that may beapplicable if the protein is subject to proteolytic cleavage. Alsoidentified were one putative casein kinase II (CKII) phosphorylationsite (at Ser¹³⁰) and four putative PKC phosphorylation sites (at Thr⁵⁴,Ser¹⁸⁰, Thr²⁰⁵ and Ser³⁷¹) (FIG. 7). These putative phosphorylationsites are also found in both murine sphingosine kinase isoforms,although the mouse enzymes also display six more possiblephosphorylation sites; four for PKC, and one each for CKII and proteinkinase A, that do not occur in hSK.

A search of signalling domain sequences using the SMART search tool(Schultz et al., 1998; Ponting et al., 1999) revealed similarity inresidues 16 to 153 of hSK to the putative diacylglycerol kinase (DGK)catalytic domain. hSK showed an overall 36% identity to the consensussequence of the DGK catalytic domain family, and possessed 17 of the 24very highly conserved amino acids of this domain. hSK, however, showedno homology with the proposed ATP binding motif of this domain(GxGxxGx_(n)K) (SEQ ID NO:8), although it should be noted that theapplicability of this protein kinase ATP-binding site motif (Hanks etal., 1988) to DGKs remains contentious (Schaap et al., 1994); Sakane etal., 1996; Masai et al., 1993). Further sequence analysis of the humansphingosine kinases also failed to find regions showing any markedsimilarity to the proposed nucleotide-binding motifs found in otherprotein families (Saraste et al., 1990; Walker et al., 1982). Apart fromthe similarity to the DGK catalytic domain, hSK shows no similarity toother lipid binding enzymes, and does not appear to have anyrecognizable lipid binding domains, like PKC C2 or pleckstrin homologydomains. There are also no other obvious regulatory domains, with thepossible exception of a proline-rich region at the C-terminus which hassome similarity to SH3 binding domains (Ren et al., 1993; Yu et al.,1994).

EXAMPLE 9 Expression in E. Coli and Isolation of Recombinant SphingosineKinase—Results

The hSK cDNA was subcloned into the pGEX 4T-2 plasmid and hSK expressedas a glutathione s-transferase (GST) fusion protein in E. coli BL21 byIPTG induction (FIG. 10). After the GST-hSK fusion protein was partiallypurified using glutathione Sepharose 4B, and the GST removed by thrombincleavage, the hSK was further purified by subsequent elutions fromcalmodulin Sepharose and Mono-Q anion exchange columns. This resulted inhigh recovery of sphingosine kinase activity (greater than 70% of theoriginally induced activity), and an electrophoretically puresphingosine kinase (FIG. 10). However, only low protein yields of therecombinant enzyme could be obtained since a large proportion of theIPTG induced, thrombin-cleaved hSK protein did not bind to thecalmodulin Sepharose column (FIG. 11). This non-binding form of hSK hadno demonstrable catalytic activity, suggesting that it was incorrectlyfolded.

EXAMPLE 10 Post-Translational Modification Requirement for SphingosineKinase Functional Activity

To determine if post-translational modifications are required foractivity of the native sphingosine kinase, the native molecule has beencompared to the recombinant enzyme produced in E. coli where suchmodifications would not occur. Specifically, the enzymes have beenexamined for differences in substrate affinity and accessibility. Thepremise for this study was that post-translational modifications maycause conformational changes in the structure of sphingosine kinasewhich may result in detectable changes in the physico-chemical orcatalytic properties of the enzyme. In summary, it was determined thatrecombinantly produced sphingosine kinase retains its functionalactivity even in the absence of post-translational modification.

Methods

Substrate Specificity of the Native and Recombinant Sphingosine Kinases

Relative rates of phosphorylation of sphingosine by the native andrecombinant sphingosine kinases were arbitrarily set at 100% andcorrespond to 2.65 kU and 7.43 kU of the native and recombinantsphingosine kinases, respective. The substrates examined were added to afinal concentration of 100 μM in 0.25% (w/v) Triton X-100, and assayedunder the standard assay conditions outlined earlier.

Substrate and Inhibitor Kinetics of the Native and RecombinantSphingosine Kinases

Substrate kinetics were determined by supplying substrates over theconcentration range of 0.5 to 200 μM for sphingosine analogues, and 5 to1000 μM for ATP. Inhibition kinetics were determined by the use ofinhibitors over a concentration range of 2 to 50 μM (Table 5). In bothcases the data were analyzed by non-linear regression.

Thermal Stabilities of the Native and Recombinant Sphingosine Kinases

Thermal stabilities of the native and recombinant sphingosine kinaseswere determined by assaying the residual activity remaining afterpreincubation of the enzymes at various temperatures (4 to 80° C.) for30 min at pH 7.4 (50 mM Tris/HCl containing 10% glycerol, 0.5M NaCl and0.05% Triton X-100). The original activities of the native andrecombinant sphingosine kinases were arbitrarily set at 100% andcorrespond to 2.65 kU and 7.43 kU, respectively.

pH Stabilities of the Native and Recombinant Sphingosine Kinases

pH stabilities of the native and recombinant sphingosine kinases weredetermined by assaying the residual activity remaining afterpreincubation of the enzymes at various pH's at 4° C. for 5 hr. Theoriginal activities of the native and recombinant sphingosine kinaseswere arbitrarily set at 100% and correspond to 2.65 kU and 7.43 kU,respectively.

The Effect of pH on Activity of the Native and Recombinant SphingosineKinases

The effect of pH on the activity of the native and recombinantsphingosine kinases were determined by assaying the activity over the pHrange of 4 to 1 in 50 mM buffers (sodium acetate, pH 4.0-5.0; Mes, pH6.0-7.0; Hepes, pH 7.0-8.2; Tris, pH 8.2-10.0; Caps, pH 10.0-11.0). Themaximum activities of the native and recombinant sphingosine kinaseswere arbitrarily set at 100% and correspond to 2.65 kU and 7.43 kU,respectively.

Effect of Metal Ions on the Activity of the Native and RecombinantSphingosine Kinases

The effect of metal ions on the activity of the native and recombinantsphingosine kinases were determined by assaying the activity understandard conditions in the presence of various metal ions or EDTA at 10mM. The maximum activities of the native and recombinant sphingosinekinases were arbitrarily set at 100% and correspond to 2.65 kU and 7.43kU, respectively.

Effect of Phospholipids on the Activity of the Native and RecombinantSphingosine Kinases

The effect of various phospolipids on the activity of the native andrecombinant sphingosine kinases were determined by assaying the activityunder standard conditions in the presence of these phospholipids at 10mol % of Triton X-100. The activities of the native and recombinantsphingosine kinases in the absence of phospholipids were arbitrarily setat 100% and correspond to 2.65 kU and 7.43 kU, respectively. PC,phosphatidylcholine; PS phosphatidylserine; PE,phosphatidylethanolamine; PI, phosphatidylinositol.

Results

Maximum activity of the native and recombinant sphingosine kinases wereobserved at pH 7.4, with both enzymes showing greater than 60% ofmaximum activity in the pH range 6.8 to 7.4 (FIG. 12). Both sphingosinekinases retained more than 90% of the original activity after 5 hincubation at 4° C. in the pH range 6 to 7.8 (FIG. 12), and at pH 7.4 inthe presence of 10% glycerol, 0.5M NaCl and 0.05% Triton X-100, bothenzymes were stable for 30 min at temperatures up to 37° C. (FIG. 12).The enzymes were much less stable in buffers lacking glycerol, NaCl andTriton X-100 (data not shown), consistent with previous observations ofthe bovine brain and rat kidney sphingosine kinases (Louie et al., 1976;Olivera et al., 1998). Both human sphingosine kinases showed arequirement for divalent metal ions since the presence of EDTA in assayselimated activity (FIG. 12). Like other sphingosine kinases examined(Louie et al., 1976; Buehrer & Bell, 1992; 1993; Olivera et al., 1998;Nagiec et al., 1998), both human enzymes showed highest activity withMg²⁺, somewhat lower activity with Mn²⁺, and only very low activity withCa²⁺. Other divalent metal ions examined, including ZN²⁺, Cu²⁺ and Fe²⁺,did not support sphingosine kinase activity (FIG. 12).

The native and recombinant sphingosine kinases have very similar, andnarrow, substrate specificites (FIG. 13), with both showing greatestactivity with the naturally occurring mammalian substrateD-erythro-sphingosine as well as D-erhthro-dihydrosphingosine. Lowactivity was also detected for both enzymes against phytosphingosine,while a range of other sphingosine derivatives and related moleculeswhere not phosphorylated. These included DL-threo-dihydrosphingosine,N,N-dimethylsphingosine, N,N,N-trimethylsphingosine, N-acetylsphingosine(C₂-ceramide), diacylglycerol (1,2-dioctanoyl-sn-glycerol and1,2-dioleoyl-sn-glycerol), and phosphatidylinositol. Further analysis ofthe human sphingosine kinases with D-erythro-sphingosine as well asD-erhthro-dihydrosphingosine revealed Michaelis-Menten kinetics over theconcentration range used (FIG. 13), with both isolated native andrecombinant sphingosine kinases showing very similar kinetic propertiesand slightly higher affinity for D-erythro-sphingosine as well asD-erhthro-dihydrosphingosine (Table 5). Both enzymes also displayedsimilar kinetics when sphingosine was supplied as a sphingosine-BSAcomplex, although presentation of the substrate in this manner resultedin a lower k_(cat) values for both enzymes (28 s⁻¹ and 39 s⁻¹ for thenative and recombinant sphingosine kinases, respectively) compared toits presentation in Triton X-100 mixed micelles, as used for all theother assays performed in this study. Sphingosine supplied as a BSAcomplex K_(m) values of 16±4 mM and 17±2 mM for the native andrecombinant sphingosine kinases, respectively. Both the native andrecombinant sphingosine kinases had the same affinity for ATP (K_(m) ofapprox. 80 mM (Table 5).

Both the native and recombinant sphingosine kinase were inhibited byDL-threo-dihydrosphingosine, N,N-dimethylsphingosine andN,N,N-trimethylsphingosine (FIG. 13), with all three of these moleculesdisplaying competitive inhibition with respect to sphingosine. Althoughthe inhibition constants for these molecules were quite similar,N,N,N-trimethylsphingosine gave slightly more efficient inhibition thanDL-threo-dihydrosphingosine, which was a marginally more efficientinhibitor than N,N-dimethylsphingosine (Table 5).

ADP also showed weak competitive inhibition, with respect to ATP (Table5). In all cases remarkably similar inhibition constants were observedfor the native and recombinant sphingosine kinases (Table 5). Noinhibition was seen with N-acetylsphingosine or Fumosin B1, a ceramidesynthase inhibitor.

The effect of calcium/calmodulin on sphingosine kinase activity wasexamined. Under the assay conditions used, calcium/calmodulin had noeffect on the activity of either the native or recombinant humansphingosine kinases (data not shown). While this result indicates a lackof sphingosine kinase activity regulation by calmodulin, the possibilityremains that calmodulin may be involved in some other function withsphingosine kinase, such as regulating ists subcellular localization.

Stimulation of sphingosine kinase activity by acidic phospholipids wasexamined. The addition of the neutral phospholipids phosphatidylcholineand phosphatidylethanolamine to the enzyme assay mixture did not resultin any detectable differences in human sphingosine kinase activity,however, marked increases in activity (1.6 to 2-fold) were observed withthe acidic phospholipids physphatidylserine, phosphatidylinositol andphosphatidic acid (FIG. 14). Both the native and recombinant sphingosinekinases were activated in a similar manner by these acidicphospholipids, with kinetic analyses revealing that all threephospholipids caused an increase in the enzymes K_(cat), while the K_(m)values remained unchanged.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

Table 3—Comparison of Sphingosine Kinase Activity in Human Placenta withVarious Animal Tissues

Fresh animal tissues were washed and homogenized as described for thehuman placenta. The proportion of cytosolic sphingosine kinase activitywas determined by comparison of the total activity in the homogenate tothat from the ultracentrifugation supernatant (100,000×g, 60 min). Thespecific activity of sphingosine kinase is expressed as pmol of S1Pformed per minute (U) per g tissue. TABLE 4 Purification of sphingosinekinase from human placenta Specific activity Cytosolic (U/g tissue) (%)Human placenta 13 51 Rat kidney 28 63 Rat liver 19 37 Rat brain 13 52Sheep kidney 38 58 Sheep liver 17 31 Sheep brain 16 63 Sheep spleen 9 34

Sphingosine kinase was purified from 1240 g of fresh human placenta (4placentas). One unit (U) of sphingosine kinase activity is defined as 1pmol of S1P formed from sphingosine and ATP per minute. TABLE 5Substrate and inhibition kinetics of the native and recombinantsphingosine kinases Specific Activity Protein Activity RecoveryPurification Step (U × 10³) (mg) (U/mg) (%) Fold Soluble fraction ofhomogenate 7943 123600 58 100 Ammonium sulphate (25-35%) 7723 3527 196697 33 Q Sepharose anion exchange 4048 1098 4597 63 79 CalmodulinSepharose 3197 18.23 1.75 × 10⁵ 40 3.0 × 10³ Mono Q anion exchange 17062.921 5.84 × 10⁵ 21.5 1.0 × 10⁴ ATP- Mono Q anion exchange 1133 0.4192.70 × 10⁶ 14.3 4.6 × 10⁴ Superdex 75 gel filtration 549 0.008 6.64 ×10⁷ 6.9 1.1 × 10⁶

Substrate kinetics were determined by supplying substrates over theconcentration range of 0.5 to 200 μM (0.0125 to 5 mol %) for sphingosineanalogues, and 5 to 1000 μM for ATP. Inhibition kinetics were determinedby the use of inhibitors over a concentration range of 2 to 50 μM (0.05to 1.25 mol %) for sphingosine analogues and 0.1 to 5 mM for ADP. Forcomparison to previous studies, all K_(m) and K_(i) values forsphingosine and its derivatives are expressed as both bulk solutionconcentrations and mol % of Triton X-100, where Triton X-100 was presentin all assays at a final concentration of 0.25% (w/v). All kineticvalues and standard errors (Duggleby, 1981) were obtained fromnon-linear regression analysis (Easterby, 1996). Native SK RecombinantSK SUBSTRATE KINETICS: Sphingosine K_(m)(mol %) 0.35 ± 0.05 0.30 ± 0.07−K_(m)(μM) 14 ± 2  12 ± 3  k_(cat)(S⁻¹) 50 85 k_(cat)/K_(m) (10⁻⁵ s⁻¹ ·M⁻¹) 36 71 Dihydrosphingosine K_(m) (mol %) 0.50 ± 0.05 0.48 ± 0.05K_(m) (μM) 20 ± 2  19 ± 2  k_(cat) (S⁻¹) 35 76 k_(cat)/K_(m) (10⁻⁵ s⁻¹ ·M⁻¹) 18 39 ATP K_(m) (μM) 77 ± 11 86 ± 12 INHIBITOR KINETICS:N,N-dimethylsphingosine K_(i) (mol %) 0.20 ± 0.03 0.19 ± 0.02 K_(i) (μM)7.8 ± 1   7.5 ± 1   DL-threo-dihydrosphingosine K_(i) (mol %) 0.15 ±0.02 0.14 ± 0.03 K_(i) (μM) 5.9 ± 1   5.7 ± 1  N,N,N-trimethylsphingosine K_(i) (mol %) 0.12 ± 0.03 0.10 ± 0.02 K_(i)(μm) 4.6 ± 1   3.8 ± 1   ADP K_(i) (mM) 1.1 ± 0.3 0.9 ± 0.2

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1. A method of modulating the functional activity of sphingosine kinasein a mammal, said method comprising administering to said mammal amodulating effective amount of an agent for a time and under conditionssufficient to increase or decrease sphingosine kinase activity.
 2. Amethod of modulating cellular function activity in a mammal said methodcomprising administering to said mammal an effective amount of an agentfor a time under conditions sufficient to modulate the expression ofsphingosine kinase or sufficient to modulate the activity of sphingosinekinase.
 3. A method of modulating cellular functional activity in amammal, said method comprising administering to said mammal an effectiveamount of a sphingosine kinase protein or a derivative, analogue,chemical equivalent or mimetic thereof, for a time and under conditionssufficient to modulate the functional activity of said cell, saidprotein optionally being a human sphingosine kinase protein; orcomprising an amino acid sequence substantially as set forth in <400>2or a derivative or mimetic thereof or a sequence having at least about45% similarity to at least 10 contiguous amino acids in <400>2 or aderivative, analogue, chemical equivalent or mimetic or said protein; orbeing encoded by a nucleotide sequence substantially as set forth in<400>1 or a derivative or analogue thereof or capable of hybridizing to<400>1 or <400>2 under low stringency conditions or a derivative,analogue, chemical equivalent or mimetic or said protein.
 4. A method ofmodulating cellular function activity in a mammal, said methodcomprising administering to said mammal an effective amount of anisolated nucleic acid molecule or derivative or analogue thereofcomprising a nucleotide sequence encoding or complementary to a sequenceencoding a novel sphingosine kinase protein or a derivative of mimeticof said sphingosine kinase protein, or a derivative, analogue, chemicalequivalent or mimetic thereof for a time and under conditions sufficientto modulate the functional activity of said cell, said nucleic acidmolecule optionally being a human sphingosine kinase protein; orcomprising a nucleotide sequence encoding, or a nucleotide sequencecomplementary to a nucleotide sequence encoding, an amino acid sequencesubstantially as set forth in <400>2 or a derivative or mimetic thereofor having at least about 45% or greater similarity to at least 10contiguous amino acids in <400>2; or comprising a nucleotide sequencesubstantially as set forth in <400>1 or a derivative thereof capable ofhybridizing to <400>1 under low stringency conditions; or which furtherencodes an amino acid sequence corresponding to an amino acid sequenceset forth in <400>2 or a sequence having at least about 45% similarityto at least 10 contiguous amino acids in <400>2, or <400>1.
 5. A methodof modulating cellular functional activity in a mammal said methodcomprising administering to said mammal an effective amount of an agentfor a time and under conditions sufficient to modulate the expression ofsphingosine kinase or sufficient to modulate the activity of sphingosinekinase wherein said sphingosine kinase expression product or sphingosinekinase modulates the activity of said cell.
 6. A method of treating amammal said method comprising administering to said mammal an effectiveamount of an agent for a time and under conditions sufficient tomodulate the expression of sphingosine kinase wherein said modulationresults in modulation of cellular functional activity.
 7. A method oftreating a mammal said method comprising administering to said mammal aneffective amount of an agent for a time and under conditions sufficientto modulate the activity of sphingosine kinase wherein said modulationresults in modulation of cellular functional activity.
 8. A method oftreating a mammal, said method comprising administering to said mammalan effective amount of a protein as recited in claim 15 or a derivative,analogue, chemical equivalent or mimetic thereof for a time and underconditions sufficient to modulate cellular functional activity.
 9. Amethod of treating a mammal said method comprising administering to saidmammal an effective amount of a nucleic acid molecule as recited inclaim 16 or a derivative, analogue, chemical equivalent or mimeticthereof for a time and under conditions sufficient to modulate cellularfunctional activity.
 10. An agent for use in modulating sphingosinekinase activity or a derivative, analogue chemical equivalent or mimeticthereof wherein modulating said sphingosine kinase activity modulatescellular functional activity; or an agent for use in modulatingsphingosine kinase expression or a derivative, analogue, chemicalequivalent or mimetic thereof wherein modulating expression of saidsphingosine kinase modulates cellular functional activity.
 11. Anisolated antibody directed to the protein recited in claim
 15. 12. Anisolated antibody directed to the nucleic acid molecule recited in claim16.
 13. The antibody according to claim 12 wherein said antibody ismonoclonal antibody.
 14. The antibody according to claim 11 wherein saidantibody is monoclonal antibody.
 15. The antibody according to claim 11wherein said antibody is a polyclonal antibody.
 16. The antibodyaccording to claim 12 wherein said antibody is a polyclonal antibody.17. A method of diagnosing or monitoring a mammalian disease conditionsaid method comprising screening for sphingosine kinase or sphingosinekinase in a biological sample isolated from said mammal.
 18. An isolatedpolypeptide, the polypeptide comprising at least 30 contiguous aminoacid residues of amino acid residues 1 to 105 of SEQ ID NO:
 2. 19. Anisolated polypeptide, the polypeptide comprising at least 50 contiguousamino acid residues of amino acid residues 1 to 105 of SEQ ID NO:
 2. 20.A composition comprising the polypeptide of claim 18, together with oneor more pharmaceutically acceptable carriers and/or diluents.